Substance-P is present in a subset of thyrotrophs in the human pituitary. Substance-P immunoreactivity and tachykinin-like peptides are present in the pituitary gland of several mammalian species. In humans, however, the biochemical nature and cellular localization of pituitary substance-P has not been defined. We report here that substance-P-immunoreactive material is present in low concentrations in both the anterior and posterior lobes of the human pituitary gland. Gel chromatography and reverse phase high performance liquid chromatography indicate that the majority of the substance-P immunoreactivity in human pituitaries elutes as authentic substance-P and its oxidized derivative. Immunohistochemical studies showed substance-P-immunoreactive fibers and terminals in the posterior pituitary gland and occasional substance-P-immunoreactive cell bodies in the anterior lobe. The substance-P-immunoreactive cells were found to colocalize with a small subpopulation of TSH beta-immunoreactive cells (thyrotrophs). Substance-P immunoreactivity was also found in a pituitary microadenoma that contained numerous TSH beta-immunoreactive cells. These studies indicate that substance-P is present in the human pituitary gland, and they suggest a relationship between substance-P and thyroid function. 