Hormonal conditions that elicit lordosis in female rats are ineffective in males, suggesting that this behavior is actively suppressed in males. Previous studies theorize that serotonergic and GABAergic inputs to the ventrolateral division of the ventromedial nucleus of the hypothalamus (VMNvl) may contribute to lordosis inhibition in males. Using triple-label immunofluorescent techniques, the present studies explored potential sex differences in the density of these projections within three hypothalamic sites: the VMNvl, the arcuate nucleus (ARC), and the dorsomedial nucleus of the hypothalamus (DMN). Antibodies directed against HuC/D, estrogen receptor alpha (ER) and either serotonin (5-HT) or the GABA synthetic enzyme GAD65 were used to compare the densities of GAD65- and 5-HT- containing fibers in each brain area, the percentage of VMNvl HuC/D immunoreactive (ir) neurons that contained ER, and the percentage of HuC/D and ER double labeled cells receiving apparent contacts from 5-HT fibers between adult, gonadectomized male and female rats. The densities of VMNvl and ARC 5-HT immunolabeled fibers were significantly higher in the males, and the percentage of VMNvl HuC/D-ir neurons containing ER was significantly higher in the females. The percentage of HuC/D-ir cells contacted by 5-HT fibers was significantly higher in the males compared to the females, but there was no sex difference in the proportion of those cells receiving contacts that were ER-ir. Neonatal administration of estradiol (E2) but not genistein (GEN) masculinized 5-HT content in the adult female VMNvl, but the percentage of HuC/D-ir cells co-labeled with ER was not significantly affected by treatment. A similar, but not statistically significant, pattern was observed in the ARC. These findings suggest that the development of serotonergic inputs to the male VMNvl is orchestrated by neonatal E2 exposure. The hormone-dependent organization of these 5-HT projection patterns may be an important developmental mechanism accounting for sex-specific behaviors in adulthood.