Substance P (SP) immunoreactivity is detectable in the rat pituitary by RIA; however, immunolocalization has been difficult. We used a sensitive immunogold silver-enhancement staining technique to cytochemically locate SP in the gland. SP-immunoreactive (SP-ir) cells were seen in anterior pituitary (AP), and occasional SP-ir fibers and terminals were seen in both AP and posterior pituitary. Colocalization studies showed the vast majority of SP-ir cells in the male AP to be also immunoreactive for growth hormone (GH). These GH/SP-ir cells represent approximately 23% of the somatotroph population in the male. SP-ir cells did not colocalize with lactotrophs, gonadotrophs, or corticotrophs; however, rare thyroid-stimulating hormone/SP-ir cells were found in the male AP. Comparisons of pituitaries from males and females revealed that females have 70% fewer SP-ir cells and that only approximately 6% of the somatotrophs in the female express SP. This sexual dimorphism is diminished in 6-day ovariectomized rats because this treatment increases the GH/SP-ir cell population 3-fold. This result suggests that the previously reported estrogen-induced decrease in SP gene and peptide expression in the pituitary occurs, at least in part, in a subpopulation of somatotrophs. To test this hypothesis, distribution of SP-ir cells was examined in pituitaries from estrogen- and oil-treated ovariectomized rats. Estrogen reduced the percentage of somatotrophs with SP immunoreactivity by 70% compared with ovariectomized oil-treated controls, indicating that estrogen most likely regulates SP levels in the pituitary by acting on a subpopulation of somatotrophs to suppress SP expression. Estrogen does not appear to alter SP immunoreactivity that is detected in the additional population of SP cells that colocalize with thyroid-stimulating hormone. These SP-expressing thyrotrophs were seen 6-fold more frequently in the female than in the male pituitary, regardless of steroid status. These studies reveal that males have more total SP-ir cells in the AP than do females and that there is a sexually dimorphic pattern of SP distribution in the gland. Males have a higher percentage of SP-ir GH cells, whereas females have more SP-ir thyrotrophs than do males. Identification of independently regulated SP-ir somatotroph and thyrotroph populations provides a basis for investigating the roles of SP in autocrine or paracrine regulation of pituitary hormone secretion.