One tenable hypothesis for the etiology of the development of prolactin-secreting adenomas is that a decrease in inhibitory dopaminergic regulation leads to increased lactotroph proliferation. Dopamine receptors have been repeatedly characterized on prolactin-secreting adenomas using labelled antagonists as ligands; however, no data are available on characterization of the receptor with a dopaminergic agonist. An agonist was utilized as the radioligand in the present study to permit the direct comparison of the pharmacological characteristics of the binding site with the biological response, the inhibition of prolactin secretion. This comparison has never been reported in tissues from the same species. Binding of the dopamine agonist and α-adrenergic antagonist [3H]-dihydroergocryptine ([3H]-DHE) to particulate fractions of surgically resected human prolactin-secreting adenomas was high affinity, monophasic, and saturable. Careful characterization of the [3H]-DHE binding by competitions with a large number of dopaminergic and α-adrenergic agents revealed the presence of both dopaminergic and α-adrenergic binding sites. The presence of a saturable, high affinity α-adrenergic binding site was confirmed with the specific α-adrenergic antagonist [3H]-WB4101 as a radioligand. Although the rank order of potency for dopaminergic compounds to compete for [3H]-DHE binding was consistent with an interaction with a dopamine receptor, the inhibitory constants (Ki) calculated from the competitions were higher than expected at an anterior pituitary dopamine receptor. This appeared to be due to the lower affinity of these agents at the α-adrenergic sites. The observed potency of dopaminergic compounds was inversely related to the number of α-adrenergic sites. The concentration of [3H]-DHE necessary to occupy half of the dopamine receptors (0.75 nM) on the particulate fractions was in close agreement with the amount of this compound which inhibits 50% of the suppressible prolactin secretion from cultured adenomas (0.35 nM). We conclude that prolactin-secreting adenomas contain a high affinity dopamine receptor which interacts with both agonists and antagonists in a similar fashion as reported for the anterior pituitary of several species. Atypically these tumors contain relatively large numbers of α-adrenergic sites which do not appear to be involved in the regulation of prolactin se