Hypothalamic catecholaminergic influences on prolactin release were investigated in vitro. Both dopamine and norepinephrine caused long lasting inhibition of prolactin release from either an isolated hemipituitary or a hemipituitary coincubated with a hypothalamus. Epinephrine also inhibited prolactin release. L-Dihydroxyphenylalanine (L-dopa) inhibited prolactin release from pituitaries in the presence of a hypothalamus but not in isolated pituitaries. DL-Threodihydroxyphenylserine (threodops), serotonin, 5-hydroxy-L-tryptophan (5-HTP), tyramine, octopamine, synephrine, thyrotropin-releasing hormone (TRH), luteinizing hormone releasing hormone (LH-RH), and somatostatin all failed to alter prolactin release. Results confirm that dopamine and norepinephrine directly inhibit prolactin release from pituitary and suggest that the hypothalamic mechanism inhibiting prolactin involves dopamine but not norepinephrine.