We investigated whether the inhibition of prolactin secretion from pituitary cells by carbachol, a cholinergic agonist resistant to hydrolysis by cholinesterases, would be a useful bioassay to explore an important nonneuronal action of antimuscarinic agents. Carbachol inhibited prolactin secretion from cultured rat anterior pituitary cells in a dose-dependent manner with a mean IC50 of 1.5 +/- 0.6 (S.E.) microM and maximal inhibition at 10(-5) M. Prolactin levels in media were significantly reduced by 30 min of incubation with carbachol. This inhibition persisted for 24 hr and was reversed by 2 microM atropine. The stimulation of prolactin secretion by 10(-6) M thyrotropin releasing hormone (to 1.5 times control) was inhibited by the addition of carbachol (10(-5) M). Addition of atropine (2 microM) to these agents restored maximal stimulation by thyrotropin releasing hormone. The inhibition of carbachol by atropine was competitive, whereas the inhibition of thyrotropin releasing hormone by carbachol was noncompetitive. The apparent affinities (Ki) of several antimuscarinic agents in pituitary cells were determined by their ability to reverse the carbachol inhibition of prolactin secretion: atropine 0.14 nM, scopolamine 0.26 nM, azaprophen 0.3 nM, aprophen 3.0 nM, pirenzepine 42 nM, benactyzine 80 nM and adiphenine 198 nM. These potencies correlated positively with those previously determined for inhibition of alpha amylase secretion from pancreatic acinar cells as well as with those for behavioral depressant actions of the antimuscarinics. Cultured anterior pituitary cells thus provide an effective system for testing the relative potencies of muscarinic antagonists in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)