The action of repaglinide, a novel insulin secretagogue, was compared with the sulfonylurea glibenclamide with regard to the hypoglycemic action in vivo, binding to betaTC-3 cells, insulin secretion from perifused mouse islets, and capacity to stimulate exocytosis by direct interaction with the secretory machinery in single voltage-clamped mouse beta-cells. Two binding sites were identified: a high-affinity repaglinide (KD = 3.6 nmol/l) site having lower affinity for glibenclamide (14.4 nmol/l) and one high-affinity glibenclamide (25 nmol/l) site having lower affinity for repaglinide (550 nmol/l). In contrast to glibenclamide, repaglinide (in concentrations as high as 5 micromol/l) lacked the ability to enhance exocytosis in voltage-clamped beta-cells. Repaglinide was more potent than glibenclamide in stimulating insulin release from perifused mouse islets (EC50 29 vs. 80 nmol/l). The greater potency of repaglinide in vitro was paralleled by similar actions in vivo. The ED50 values for the hypoglycemic action were determined to be 10.4 and 15.6 microg/kg after intravenous and oral administration, respectively. The corresponding values for glibenclamide were 70.3 microg/kg (intravenous) and 203.2 microg/kg (oral). Further, repaglinide (1 mg/kg p.o.) was effective (P <0.001) as an insulin-releasing agent in a rat model (low-dose streptozotocin) of type 2 diabetes. These observations suggest that the insulinotropic actions of repaglinide and glibenclamide in vitro and in vivo are secondary to their binding to the high-affinity repaglinide site and that the insulinotropic action of repaglinide involves both distinct and common cellular mechanisms.