Small fluctuations in cholinergic input may be involved in the regulation of human interdigestive pancreatic secretion. To determine the effects of small changes in cholinergic tone on phase II interdigestive trypsin secretion, we gave cholinergic agonists or antagonists to 17 healthy fasting subjects who underwent gastrointestinal intubation. Duodenal trypsin outputs, gastroduodenal motility, and plasma levels of pancreatic polypeptide (PP) as a marker of cholinergic tone were measured during intravenous infusion of bethanechol (0, 5, or 40 micrograms.kg-1.h-1) or atropine (0, 4, or 16 micrograms.kg-1.h-1) given during phase II alone or in combination based on a 3 x 3 factorial design. Data were evaluated by a response surface analysis of the average log values for the test period (using the average of log values of the control period as a covariate). Bethanechol increased trypsin output (p < 0.05) and plasma concentrations of PP (p < 0.05) within the ranges of spontaneous fluctuations of trypsin output and PP during phase II, but did not disrupt the periodicity of pancreatic secretion or the characteristic cyclical pattern of interdigestive motility and induction of phase III activity. Atropine markedly decreased trypsin output (p < 0.05) and plasma concentrations of PP (p < 0.05) and abolished the cycling of interdigestive pancreatic secretion and motor activity. These data suggest the hypothesis that cholinergic pathways participate in the control of interdigestive pancreatic secretion during phase II, while enzyme secretion during phase III may be regulated by other mechanisms.