The effect of chronic administration of tolbutamide (150 mg/kg X day, orally, over 60 days) and glibenclamide (1 mg/kg X day, orally, over 60 days) on pancreatic A, B, and D cell function was investigated in male nondiabetic rats. In a first set of experiments pancreatic hormonal response to metabolic stimuli was evaluated during glucose (11.1 mM) or arginine (10 and 20 mM) infusion in the isolated perfused rat pancreas. The basal levels of insulin (IRI) and glucagon (IRG) were similar in control and in sulfonylurea-treated rats. Tolbutamide treatment markedly depressed the IRI response to glucose (P less than 0.005) or arginine (P less than 0.0005) infusion and the IRG response to arginine (P less than 0.01). After glibenclamide treatment, IRI decreased significantly (P less than 0.0005 and P less than 0.005, respectively) only in response to arginine infusion. This effect was still evident 10 days after the end of treatment. Furthermore, long term glibenclamide administration suppressed somatostatin (SRIF) response to glucose (P less than 0.0005) or arginine (P less than 0.0005). In a different group of rats treated with glibenclamide (1 mg/kg X day, orally, over 60 days) IRI, IRG, and SRIF plasma concentration and blood glucose levels were examined at the end of treatment. The results did not differ significantly from those of a control group. In the same animals, pancreatic IRI, IRG, and SRIF content, measured on acid-ethanol extracts, was reduced (P less than 0.1 vs. controls). These data clearly indicate that long term treatment with sulfonylurea drugs has a suppressive effect on pancreatic endocrine function in rats. The concomitant involvement of A, B, and D cell suggests that this effect is not specific.