Surface glucose transporters of intact muscles were photolabeled with the membrane impermeant ATB-[2-3H]BMPA reagent and localized by autoradiography. We found sparse labeling of the glucose transporters by ATB-[2-3H]BMPA on the sarcolemmal membrane around the muscle fiber. The majority of label was on the interior of the muscle fiber, at a discrete site which matched the distribution of AI junctions and which was presumed to be on the exterior surface of T-tubules. The amount of photolabel on the T-tubule was increased in response to insulin and was blocked by cytochalasin B. These results support the concept that glucose transport may occur predominantly across the T-tubule membrane under basal and insulin-stimulated conditions.