The effect of sulfonylureas on long-term regulation of glucose uptake by insulin and insulin-like growth factors has been studied in the L6 line of cultured skeletal muscle cells. These cells have previously been shown to possess many characteristics of differentiated skeletal muscle and to bind and respond to physiological concentrations of insulin and insulin-like growth factors I and II. Tolazamide (half-maximal at 0.2 mg/ml) augments the effects of insulin, insulin-like growth factor I, and insulin-like growth factor II on glucose uptake, increasing both sensitivity and maximal efficacy of the hormones. In the absence of added hormone, tolazamide has no effect on glucose uptake. A similar increase in insulin-stimulated glucose uptake with unaltered basal uptake occurs with glyburide (half-maximal at 0.5 microgram/ml). The action of tolazamide requires long-term exposure to the sulfonylurea (22 h) and is inhibited by cycloheximide, suggesting a process that involves new protein synthesis. In contrast to glucose uptake, amino acid uptake in L6 cells is increased by tolazamide in the absence of hormones. Insulin and the insulin-like growth factors also stimulate amino acid uptake, but this effect is not further augmented by tolazamide. Thus, sulfonylureas appear to directly modulate amino acid uptake, but to indirectly augment glucose uptake through an effect on insulin and insulin-like growth factor stimulated pathways. Neither insulin binding nor insulin degradation is altered by tolazamide, indicating a post-binding mechanism of action. The L6 cultured skeletal muscle cell line should be useful in future studies on the mechanism of the extrapancreatic actions of sulfonylureas.