The sulfonylurea glyburide, a 'second-generation' oral hypoglycemic compound, was studied in vitro in order to determine its cellular mechanism of action in adipocytes prepared from cultured rat epididymal fat tissue. Glyburide treatment (1 microgram/ml) for 20 h did not alter insulin receptor number or affinity, or down-regulation by insulin. Biologic responses of these cells were measured in the presence of insulin or the oxidants Vitamin K5 and H2O2, which have insulin-like activity, but do not act through the binding portion of the receptor. 2-Deoxyglucose uptake was not significantly changed by exposure to glyburide alone. However, the sulfonylurea increased the insulin-stimulated or insulin-mimicker-activated uptake by approximately 30%. Insulin-stimulated glucose oxidation was also potentiated when glucose transport was rate limiting for metabolism. These findings extend our earlier observation that in adipose tissue the primary cellular mechanism of action of sulfonylureas is to potentiate insulin-stimulated hexose transport, and that this process may account for their hypoglycemic activity.