In the present study, we have used 125I-cyanopindolol (125ICYP) to identify, characterize, and localize beta-adrenergic receptors in bovine, rat, and human pituitary gland by in vitro labeling light microscopic autoradiography. The binding of 125ICYP to slide-mounted bovine pituitary sections was saturable and of high affinity with an apparent Kd of 0.2 nM. The pharmacological profile of 125ICYP binding obtained from competition studies demonstrates that the beta-adrenergic receptors in the pituitary gland are predominantly of the beta 2 subtype. Rat pituitary autoradiograms show specific binding sites for 125ICYP in anterior, intermediate, and posterior lobes with highest concentrations found in the intermediate lobe and progressively lower concentrations in posterior and anterior lobes, respectively. Autoradiograms of 125ICYP binding in human pituitary show a significantly higher concentration of beta 2-adrenergic receptors in posterior than in anterior lobe of the pituitary. There is a homogeneous distribution of beta 2-adrenergic receptors within each lobe of both rat and human pituitary glands. The results of the present study provide the first visualization of beta 2-adrenergic receptors in rat and human pituitary and demonstrate the presence of significant concentrations of beta 2-adrenergic receptors in the posterior lobe. The data support a role for epinephrine and norepinephrine in modulating pituitary function.