In this study on purified rat pancreatic β-cells, we show that the second-generation sulfonylurea glyburide stimulates insulin release through a direct interaction with the β-cells. During static incubations, 2 μM glyburide releases 0.16 pg insulin per β-cell, which corresponds to a half-maximal glucose stimulation. This effect occurs independently from the glucose-recognition unit, being detectable at both nonstimulatory and stimulatory glucose concentrations and proceeding without alterations in the rate of glucose oxidation. The secretagogue action of glyburide appears not to be mediated through cAMP but is potentiated by cAMP-generating substances such as glucagon (10⁻⁸ M; 0.31 pg insulin released per β-cell). Its 10-fold higher potency in isolated islets is attributed to the markedly higher cAMP levels that are maintained in islet β-cells under the influence of locally released glucagon. Perifused pancreatic β-cells respond to glyburide with a biphasic insulin release. After removal of the drug, the cells continue to secrete insulin at the same rate for ≥ 30 min. This prolonged secretory activity coincides with a cellular accumulation of the drug, primarily in association with membranes of secretory vesicles and mitochondria. Tolbutamide also stimulates insulin release from pure β-cells, but it is less powerful on a molar basis and does not lead to a sustained hormone release after its removal from the extracellular medium. We conclude that the hypoglycemic action of glyburide is at least partly the result of a direct interaction with pancreatic β-cells. The insulin-releasing action of the drug has been shown to occur independently of both the nutrient-dependent and the cAMP-generating regulatory units but to vary markedly with the cellular cAMP content and with the prolonged and possibly intracellular actions of the drug.