Substance P (SP) is known to act on pituitary hormone release either at the hypothalamic level or directly at the pituitary level. In order to investigate whether SP is present in the pituitary gland and to localize the peptide at the cellular and subcellular levels, the immunocytological method was used. Rat pituitaries were fixed and frozen. Ultrathin sections, obtained by cryoultramicrotomy, were incubated with anti-SP serum. The antigen-antibody reaction was detected by 4-chloro-1-naphtol. SP immunoreactivity was observed both in the prolactin and in the gonadotropic cells, but not in the somatotropic, corticotropic and thyrotropic cells. In reactive cells, SP immunoreactivity was observed in the secretory granules, in the cytoplasm, and in the nucleus distributed all over the euchromatin near to the heterochromatin regions. No immunoreactivity was observed when nonimmune serum or anti-SP serum incubated with SP was used. No modification of the immunocytochemical reaction was observed when anti-SP serum incubated with somatostatin, gonado- or thyroliberin was used. These data (1) provide immunocytological evidence for the presence of SP in the pituitary gland; (2) indicate the presence of SP peptide in the gonadotropic and prolactin cells only. They support previous findings indicating that SP could have a direct effect at the pituitary level.