The plant alkaloid, catharanthine, was shown to stimulate release of amylase from pancreatic fragments and to cause extensive degranulation of pancreatic acinar cells with accumulation of membrane material in the Golgi region. The extent and time course of maximal catharanthine stimulation was comparable to that induced by the cholinergic analog bethanechol. Antimycin inhibited the action of catharanthine while atropine did not. Removal of Ca2+ from the incubation medium inhibited amylase release induced by catharanthine but did not affect release induced by bethanechol. Catharanthine induced a delayed release of 45Ca2+ from prelabeled pancreatic fragments as compared to bethanechol. It is suggested therefore that catharanthine activates the physiological pathway controlling amylase release by causing a rise in cytoplasmic Ca2+ but the mechanism by which this occurs is different from that caused by physiological secretagogues.