Steady-state concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in anterior hypothalamus (AH), preoptic area (POA), suprachiasmatic nucleus (SCN), and ventromedial nucleus (VMH) were equivalent 30 days after castration or 17β (E2) or E2 + DHT for 30 days to castrated males also failed to alter differentially the concentrations of 5-HT and 5-HIAA in any of the brain regions assayed. Although castrated and sham-operated rats had similar levels of 5-HT and 5-HIAA in whole hypothalamus + preoptic area (H + POA), brain stem (BS), and cortex (CTX) when measured 7 and 32 days postoperatively, the mounting rates and the percentage of animals ejaculating were similar for both groups 6 days postoperatively whereas only the sham-operated animals continued to mount at a high rate and to ejaculate 31 days postoperatively. Also, 30 days after surgery, there were no differences among groups of males which were either castrated, castrated and given a subcutaneous capsule of testosterone, or sham operated, in the accumulation of 5-hydroxytryptophan in the H + POA, BS, or CTX measured 30 min after an intraperitoneal injection of an aromatic amino acid decarboxylase inhibitor. These results suggest that the hormonal activation of masculine sexual behavior in the rat is not mediated by a change in the metabolic activity of 5-HT neurons in either the hypothalamus or POA