Brief exposure (30 min) of isolated islets to 0.5 microgram/ml cyclosporine leads to alterations in the insulin secretory response to selected stimuli. When glucose is used as the secretagogue, cyclosporine slightly stimulates insulin release at substimulatory concentrations of the hexose. The inhibitory effect predominates, however, at stimulatory concentrations of glucose with a threshold at 6 mmol/L glucose and maximal inhibition of 33.5 mmol/L glucose. By contrast, insulin release induced by 17 mmol/L arginine is not affected. Cyclosporine also inhibits by 66% the insulin secretory response to 100 nmol/L phorbol 12-myristate 13-acetate, suggesting that either cyclosporine interferes with phorbol ester action on beta cells or the action site is located beyond the protein kinase C activation. On the other hand ionomycin-stimulated insulin response is also blocked by cyclosporine, indicating that insulin release induced by transient changes in cytosolic Ca++ is also affected. The evidence gathered here suggests that the inhibitory effect of cyclosporine on insulin release is apparent when glucose is used as a fuel stimulant and is reversed following removal of the stimulant. This effect is not reversed by using substances known to activate the protein kinase C or the Ca(++)-dependent branches of the stimulus-secretion coupling system in beta cells, indicating that the site of action of cyclosporine on pancreatic islets might be located in distal steps of the stimulus-secretion coupling of glucose-induced insulin release.