3H-Dexamethasone (10 microgram/kg) was injected intravenously in adrenalectomized rats and after survival times of 5, 30, 60, and 180 min its uptake within the pituitary was studied by autoradiography. Radioactivity was concentrated in cell nuclei in the pars nervosa and pars distalis. Within the pars intermedia, only cells of the marginal zone were labeled. In the pars distalis, some cells showed a weak nuclear accumulation of radioactivity as early as 5 min after injection. The tissue radioactivity was nearly maximal at 5 min, and the proportion of radioactivity in nuclei reached a maximum of 60-70% by 30 min. In competition experiments, non-radioactive steroids (1 mg/kg) were injected 5 min before 3H-dexamethasone and sacrifice was 30 min later. Dexamethasone markedly diminished the nuclear accumulation in the pars distalis, but corticosterone and progesterone did not. In the pars nervosa, corticosterone and progesterone competed for nuclear uptake of 3H-dexamethasone, although less effectively than dexamethasone itself. Different cell types in the pars distalis were characterized by treating autoradiograms with an immuno-peroxidase bridge procedure. Cells treated with anti-ACTH 17-39 had the greatest nuclear concentration of radioactivity, and those stained with anti-TSH were least heavily labeled. Cells treated with antisera to GH, PRL, and hCG were moderately labeled.