The antihelminthic drug mebendazole (MEB) has been reported to elevate circulating insulin levels in vivo. By using isolated perifused rat islets we tested the acute effects of this drug directly on the beta cell in the absence or presence of exogenous glucose. In the absence of glucose, MEB (40 micrograms/ml) caused a delayed but sustained increase in insulin output from the islet. Release induced by this concentration of MEB was not influenced by either forskolin (10 microM), a compound that increases islet cyclic AMP levels and potentiates glucose-induced secretion, or the absence of extracellular calcium. In the presence of a submaximal stimulatory glucose concentration (8 mM), release was dramatically potentiated by the further addition of 10 to 40 micrograms/ml of MEB. The potentiating effect of the drug on glucose-induced release was readily reversible and abolished by the omission of extracellular calcium or the addition of 10 mM mannoheptulose, a compound that blocks glucose phosphorylation. Despite its positive effects on hormone secretion, MEB did not affect glucose metabolism. MEB may prove useful for investigating the factors that regulate insulin secretion.