It was recently proposed that yohimbine (YOH), an indole alkaloid with multiple pharmacological effects, is an antagonist of the D2 dopamine (DA) receptor. Because the pituitary DA receptor involved in the inhibition of prolactin (PRL) secretion is the prototypic D2 receptor, we examined the effect of YOH on PRL secretion in male rats. YOH produced marked, dose-dependent and sustained increases in plasma PRL levels. However, YOH did not block the inhibitory effect of DA on PRL release from rat pituitary glands in vitro, did not displace [3H]spiperone from bovine pituitary membranes and had no effect on the concentration of DA in pituitary stalk plasma of anesthetized rats, suggesting that the stimulation of PRL release by YOH is not due to its antidopaminergic effects. Clonidine, an alpha-2 adrenergic agonist, produced a partial, non-dose-dependent inhibition of the YOH-induced rise in serum PRL levels. Two antagonists of the H1 histamine receptor, diphenhydramine and promethazine, markedly antagonized the PRL-releasing effect of YOH, but another H1 blocker, chlorpheniramine, and an H2 antagonist, metiamide, had no effect. Serotonin receptor blockers, cyproheptadine, mianserin and pizotifen, and the opiate antagonist, naloxone, also had no effect on the PRL response to YOH. Nevertheless, the PRL-releasing effect of YOH was potentiated 24 hr after the administration of reserpine or para-chlorophenylalanine, an inhibitor of serotonin synthesis. Thus, the mechanisms by which YOH stimulates rat PRL secretion has has not been fully elucidated. It is possible that YOH may stimulate PRL secretion by a novel mechanism, possibly through the intervention of a PRL-releasing factor.