The activity of melanotroph cells of the amphibian pars intermedia is regulated by multiple factors including classical neurotransmitters and neuropeptides. In this study, we have examined the possible involvement of acetylcholine (ACh) in the regulation of electrical and secretory activities of frog pituitary melanotrophs. Electrophysiological recordings were conducted on cultured cells by using the patch-clamp technique in the whole-cell configuration. In parallel, alpha-MSH release from acutely dispersed pars intermedia cells was studied by means of the perifusion technique. In all cells tested in the current-clamp mode, superfusion with ACh (10(-6) M) gave rise to a depolarization associated with an enhanced frequency of action potentials. Administration of ACh (10(-6) M) to perifused cells also induced stimulation of alpha-MSH release. These results indicate that the neurotransmitter ACh exerts a direct stimulatory effect on pituitary melanotrophs. The action of ACh on electrical and secretory activities was mimicked by muscarine (10(-5) M), while ACh-induced alpha-MSH secretion was completely abolished by the muscarinic antagonist atropine (10(-6) M). The depolarizing effect of muscarine was suppressed by the specific M1 muscarinic antagonist pirenzepine (10(-5) M), indicating the existence of a M1 subtype muscarinic receptor in frog pars intermedia cells. In addition, using a monoclonal antibody against calf muscarinic receptors, we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in cultured intermediate lobe cells. Electrophysiological recordings showed that nicotine (10(-5) M) induces membrane depolarization associated with an increase of the frequency of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)