We investigated the influence of ouabain on glucose-induced insulin release from toad pancreatic minces in the same nanomolar range as that of an ouabain-like compound found in human blood. Ouabain increased insulin secretion at basal (2 mM), but not maximally stimulatory (8 mM), glucose levels in a dose-dependent manner up to an optimal concentration of 1 nM, with the values declining progressively thereafter. Ouabain at 3 nM changed the shape of the overall dose-response curve for glucose from sigmoidal to hyperbolic and displaced the optimal insulinotropic glucose concentration from 8 to 2 mM. Preincubation with ouabain (3 nM) followed by glycoside washout potentiated insulin induction at 2 mM, but not at 8 mM glucose, but this same pretreatment followed by incubation in Ca(2+)-free medium depressed insulin release under all conditions, and especially at high glucose; here, however, the preexposure to ouabain partially prevented the drop in insulin secretion at 8 mM glucose. Acetylcholine at 8 microM augmented insulin release at both levels of glucose, and ouabain potentiated this effect synergistically at high, but not low glucose. Ouabain, at physiologic concentrations, thus appears to regulate the effect of secretagogues such as glucose and acetylcholine in amphibians.