A single injection of pimozide, a dopamine antagonist, rapidly stimulated prolactin (PRL) gene transcription in male rats, whereas an injection of alpha-ergocryptine, a dopamine agonist, rapidly inhibited PRL gene transcription. Pretreatment with cycloheximide blocked the induction of PRL gene transcription by pimozide but had no effect on the inhibition of transcription by ergocryptine. The interactions between ergocryptine and 16 alpha-estradiol, an estrogen that stimulates PRL gene transcription through two independent mechanisms, were also examined. Pretreatment with ergocryptine had no effect on the ability of 16 alpha-estradiol to stimulate PRL gene transcription through a mechanism that is most probably mediated directly by the anterior pituitary estrogen receptor. However, ergocryptine pretreatment did block the ability of 16 alpha-estradiol to stimulate transcription through a second, indirect, mechanism. This ergot alkaloid also blocked the ability of pimozide to stimulate PRL gene transcription. Pretreatment with 16 alpha-estradiol had no effect on the ability of ergocryptine to inhibit PRL gene transcription, indicating that this estrogen did not grossly alter the responsiveness of the anterior pituitary to the dopamine agonist. The similarities between the effects of 16 alpha-estradiol, via the indirect mechanism, and pimozide on PRL gene transcription suggest that estrogen may stimulate PRL gene transcription in vivo in part by reducing the release of dopamine from hypothalamic neurons.