Atrial natriuretic factors (ANF) are a family of peptides originally identified in atrial cardiocytes and having natriuretic, diuretic and vasorelaxatory properties. ANF was recently reported to be synthesized in anterior pituitary cells. In the current study, the cell-specific sites of binding and internalization of ANF in the adenohypophysis, a site of ANF action, were investigated. By in vitro autoradiographic techniques, [125I]ANF was found to bind specifically to both anterior and posterior lobes of the pituitary, but not to the intermediate lobe. 5 min following intravenous injection of [125I]ANF, radiolabel was detected by ultrastructural autoradiography on gonadotrophs, corticotrophs and lactotrophs, but not thyrotrophs or somatotrophs. Label was found at both the plasma membrane level and cytoplasmic matrix, but not in the nuclei, of all three cell types. The cellular and subcellular localization of endogenous ANF-like immunoreactivity was also investigated. Specific immunoreactivity was also detected in gonadotrophs, corticotrophs and some lactotrophs. At the subcellular level in all three cell types, immunoreactivity was localized in the cytoplasmic matrix, on secretory granule membranes, and in mitochondria. Less dense staining was observed in nuclear euchromatin; endoplasmic reticulum and Golgi apparatus were not labelled in any of the cells. The data suggest that notwithstanding their reported ability to synthesize ANF, exogenous ANF can also bind and be internalized by adenohypophyseal gonadotrophs; the binding and presence of endogenous ANF-like immunoreactivity in corticotrophs and lactotrophs suggest that these cells may be sites of action for the atrial peptide.