The actions of sulfonylurea agents to increase peripheral glucose disposal have been classically ascribed to an ability to potentiate insulin action. However, in the BC3H-1 cultured muscle cell, tolbutamide, glipizide, and glyburide directly provoked more than a twofold increase in 2-deoxyglucose (2-DG) uptake in a dose-dependent manner in the absence of insulin. Tolbutamide (3 mM) enhanced 2-DG uptake by 130% in the presence or absence of insulin and did not significantly change insulin binding or the sensitivity of the insulin response. The onset of tolbutamide-stimulated hexose transport was seen after 30 min and reached a plateau after 12 h. Tolbutamide-stimulated glucose transport was associated with a twofold increase in the Vmax of 2-DG uptake and was completely blocked by 50 microM cytochalasin B, indicating that this action is mediated by increase in cell membrane glucose transporters. We show that sulfonylureas at therapeutic concentrations directly increase glucose transport into muscle cells. Because muscle is the major peripheral target tissue for glucose disposal, these results provide the basis for the therapeutic effect of these agents in improving peripheral glucose disposal in insulin-resistant type II (non-insulin-dependent) diabetes mellitus.