The mechanism(s) by which the oral sulfonylurea, tolazamide, exerts its extrapancreatic hypoglycemic effects was studied using rat epididymal adipose tissue maintained 20-44 h in the presence or absence of the drug. Insulin binding, hexose transport and glucose metabolism were compared in adipocytes isolated from the cultured tissue. In contrast to earlier reports that suggested that sulfonylureas alter the binding of insulin, neither receptor number nor affinity were changed by tolazamide treatment. The uptake of the glucose analogs 2-deoxyglucose and 3-0-methylglucose in the absence of insulin (i.e., basal) was also unchanged. However, exposure to tolazamide resulted in a potentiation of the stimulatory effects of insulin by approximately 30% at each hormone concentration assayed (0.4-40 ng/ml). This potentiation was dependent on the tolazamide concentration (0.003-0.30 mg/ml), with a maximal effect observed at therapeutic levels. A tolazamide analog hypoglycemic activity in vivo was found not to enhance either basal or insulin-stimulated uptake in vitro. Conversion of 0.1-5.0 mM glucose to CO2 and total lipids in the presence of insulin was also potentiated by tolazamide treatment. The inability of the drug to directly stimulate basal glucose uptake was paralleled by its lack of effect on glucose metabolism. At 50 mM glucose, where transport is no longer rate-limiting, tolazamide did not potentiate metabolism in the absence or the presence of insulin. These studies demonstrate that tolazamide in vitro alters postreceptor insulin action without influencing the receptor, and suggests insulin-stimulated hexose transport as the cellular process responsible for the hypoglycemic effect of sulfonyureas in adipose tissue.