In Brother's Keeper, Sam creates the witch-killing bullets by filling hollow-point bullets with the potion from Bobby Singer's witch-killing spell in order to threaten witch Rowena MacLeod into removing the Mark of Cain from Dean's arm. However, Rowena is aware that Sam needs her to remove the Mark and remains unthreatened by him.
In The Bad Seed, Sam arms himself with witch-killing bullets once more to ensure Rowena's compliance in breaking the Attack Dog Spell she cast on Castiel. After Rowena hesitates, Sam reminds her that the gun is loaded with witch-killing bullets. Finally, Rowena breaks the spell, but disarms Sam before making her escape.
In Love Hurts, Sam removes a gun loaded with witch-killing bullets from the Impala's trunk when he and Dean go to confront evil witch Sonja and her Qareen. Sam explains to Dean that the gun is loaded with witch-killing bullets and the two comment on needing to find a better name for it. During the confrontation with Sonja, as Melissa Harper causes a distraction, Sam is able to retrieve his gun and kill Sonja with two witch-killing bullets. He is then able to kill the Qareen and save Dean's life.
In American Nightmare, after suspecting that Wicca Beth Roberts is an evil witch killing people to get ahead, Dean arms himself with witch-killing bullets to confront her. However, after learning that Beth hates her new job, Dean realizes she is innocent and relents. Despite Dean nearly killing her, Beth ends up giving him her phone number to Sam's amazement.
In Regarding Dean, after recognizing Gideon Loughlin from pictures in hex victim Barry Gilman's office, Dean confronts him. After Gideon throws Dean with telekinesis, Dean is able to mortally wound Gideon with a witch-killing bullet and chases after the witch with the intention of finishing him off. Dean's other shots miss and Gideon is able to hit Dean with the Memory Curse before dying of his wound. While retracing Dean's steps, Sam finds the shell casing from one of Dean's bullets and instantly identifies it as a witch-killing bullet, giving them a clue as to what went on.
Later, while confronting Catriona and Boyd Loughlin, Sam arms himself with witch-killing bullets but is captured by the siblings to resurrect their dead brother. A memory-less Dean, having been brought to the scene in the Impala by Rowena, follows directions to the arsenal in the trunk where Rowena has marked both his gun and the witch-killing bullets. As Catriona tortures Rowena, Dean threatens her at gunpoint causing Catriona to scoff due to her invulnerability to bullets. After displaying Rowena's note reading "witch-killing bullets", Dean kills Catriona with a shot to the head. After Boyd comes downstairs, Dean kills him with a witch-killing bullet too after a moment of indecision over whether to shoot him or Sam.
In Twigs & Twine & Tasha Banes, Dean takes good witch Max Banes to see the Impala's arsenal. After the two commentate over Dean's grenade launcher, Dean shows him the witch-killing bullets, the weapon they actually need. Later, while the Borrower Witch is distracted by Max, Dean is able to retrieve his gun and kill the witch with a shot to the chest. This breaks her animation spell for her twig dolls, saving Sam's life.
In Various & Sundry Villains, the Winchesters arm themselves with witch-killing bullets again when facing off with the Plum sisters. During the fight, both brothers lose their guns, but Rowena retrieves Dean's and uses it to kill the zombie Mrs. Plum. Rather than using the witch-killing bullets in Dean's gun to kill the Plum sisters, Rowena casts the Attack Dog Spell on them to force the two to kill each other.
In Funeralia, the Winchesters arm themselves with witch-killing bullets upon learning that Rowena is killing again. When confronted by Sam armed with them, Rowena challenges Sam to shoot her. Sam attempts to shoot Rowena who stops the bullet with telekinesis and drops it to the ground. Rowena is shocked that Sam actually tried to shoot her.
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- Starting in American Nightmare, the Winchesters are shown to keep witch-killing bullets in the Impala's trunk in a case alongside their silver bullets. In Regarding Dean, these bullets are clearly labeled by Rowena MacLeod for the memory-less Dean.
- When Sam uses witch-killing bullets, he uses a small revolver specifically loaded with them as seen in Brother's Keeper, The Bad Seed, Love Hurts, Regarding Dean, Various & Sundry Villains and Funeralia. However, Dean simply loads his regular gun with the bullets rather than using a specially loaded one as seen in American Nightmare, Regarding Dean and Twigs & Twine & Tasha Banes.
- Unlike the spell version, the bullets do not cause the targeted witch to combust upon usage. Instead, the witch dies the same as they would from any gunshot with one even bleeding out from his wound. However, due to the magical nature of the bullets, they can kill witches invulnerable even to regular bullets as Catriona Loughlin's reaction to Dean threatening her with a gun implied.
- The power of the witch shot with the bullets is also unaffected as long as they are alive as seen when Gideon Loughlin was able curse Dean after being shot.
- The bullets were initially created to threaten Rowena MacLeod into compliance in Brother's Keeper. Ironically, her life was saved by Dean with the bullets in Regarding Dean. Rowena later used one herself to kill a zombie, albeit incidentally since she used Dean's gun to shoot the zombie in the head and the gun was loaded with the bullets at the time.
- Despite Sam inventing the witch-killing bullets, Dean has the most kills with them. Sam has only used the bullets once to kill Sonja while Dean has killed three members of the Loughlin Family and the Borrower Witch with them.
- As seen on the shell casing in Regarding Dean, the Winchesters witch-killing bullets have a W carved into them to differentiate them from their other bullets.