Serotonergic dysregulation is associated with negative affect. Plasma prolactin responses to a tryptophan enhancement challenge are used as a measure of central nervous system serotonergic activity. We examined prolactin responses to a tryptophan challenge as they relate to the personality domains of neuroticism, extraversion, openness, agreeableness, and conscientiousness. Participants were 67 volunteers. Regression models assessed peak prolactin response to intravenous tryptophan infusion as a predictor of neuroticism, extraversion, openness, agreeableness, and conscientiousness. Prolactinxgender product terms were included to examine moderation by gender. Models were adjusted for baseline levels of prolactin, age, and race. Gender moderated the association between N and prolactin level (p<.03). Higher levels of N were associated with decreased levels of prolactin responses in females, whereas the opposite was true for males. Remaining personality domains were not related to prolactin levels. Findings add to literature suggesting the serotonin system functions differently, in important ways, in males and females.