The present study investigated the role of brain serotonin activity in the mediation of masculine sexual behavior in the intact male rat. The results of previous studies suggest a dissociation of the effects of gonadal hormones on central serotonin metabolism and sexual behavior in the male rat. However, serotonin activity was measured some 24 hours after the last test of sexual activity. In the present experiment, animals were assigned to one of five groups, and were sacrificed by decapitation immediately after meeting their respective behavioral criterion. High performance liquid chromatography (HPLC) was used to measure concentrations of serotonin and its metabolite, 5-hydroxyindoleacetic acid, in the hypothalamus-preoptic area. Analyses of variance revealed no significant differences in brain serotonin activity as a function of sexual behavior. Results are discussed in the context of the role of biogenic amines in the mediation of masculine sexual behavior in the male rat.