Aggression in humans and animals has been linked to androgens and serotonin function. To further our understanding of the effect of androgens on serotonin and aggression in male macaques, we sought to manipulate circulating androgens and the activity of aromatase; and to then determine behavior and the endogenous availability of serotonin. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with (a) placebo; (b) testosterone (T); (c) T Dutasteride (5a reductase inhibitor; AvodartTM); (d) T Letrozole (nonsteroidal aromatase inhibitor; FemeraTM); (e) Flutamide ATD (androgen antagonist plus steroidal aromatase inhibitor); or (f) dihydrotestosterone (DHT) ATD (n 5/group). Behavioral observations were made during treatments. At the end of the treatment period, each animal was sedated with propofol and administered a bolus of fenfluramine (5 mg/kg). Fenfluramine causes the release of serotonin proportional to endogenous availability and in turn, serotonin stimulates the secretion of prolactin. Therefore, serum prolactin concentrations reflect endogenous serotonin. Fenfluramine significantly increased serotonin/prolactin in all groups (p <.0001). Fenfluramine-induced serotonin/prolactin in the T-treated group was significantly higher than the other groups (p <.0001). Castration partially reduced the serotonin/prolactin response and Letrozole partially blocked the effect of T. Complete inhibition of aromatase with ATD, a noncompetitive inhibitor, significantly and similarly reduced the fenfluramine-induced serotonin/prolactin response in the presence or absence of DHT. Neither aggressive behavior nor yawning (indicators of androgen activity) correlated with serotonin/prolactin, but posited aromatase activity correlated significantly with prolactin (p <.0008; r2 0.95). In summary, androgens induced aggressive behavior but they did not regulate serotonin. Altogether, the data suggest that aromatase activity supports serotonin production and that androgens increase aggression by another mechanism.