The roles of the glucose transporter isoforms, GLUT1 and GLUT4, in mediating insulin-stimulated glucose transport were investigated by stably overexpressing the transporters in L6 myoblasts. Levels of GLUT1 and GLUT4 in myoblasts from the cell lines having the highest content of these transporters were approximately 16- and 30-fold higher, respectively, than levels in nontransfected cells. The basal rate of 2-deoxy[3H]glucose uptake was severalfold higher in cells overexpressing GLUT1 than in the parent L6 myoblasts or in control cell lines that were generated by transfecting cells with expression vectors lacking transporter insert. The basal rate was not elevated in any of the lines expressing GLUT4. The net increase in 2-deoxy[3H]glucose uptake produced by insulin was larger in both the GLUT1 and GLUT4 cells than in the control cells. Insulin increased uptake in GLUT4 cells by as much as 6-fold; whereas, the fold increase over basal uptake produced by insulin in GLUT1 cells was comparable to that (2-fold) observed in the control myocytes. Thus, both GLUT1 and GLUT4 can mediate insulin-stimulated glucose transport in L6 myoblasts, although GLUT4 is needed to observe large percentage increases comparable to those observed in skeletal muscle fibers in vivo. In contrast to insulin, the protein phosphatase inhibitors, okadaic acid and calyculin A, inhibited glucose transport in cells expressing either GLUT1 or GLUT4. Calyculin A, which produced a half-maximum effect at 10 nM, was approximately 100 times more potent than okadaic acid in decreasing both basal and insulin-stimulated 2-deoxyglucose uptake. Inhibition of uptake by calyculin A was associated with a decrease in the cell surface concentration of both GLUT1 and GLUT4. These results indicate that increased protein phosphorylation can lead to inhibition of transport mediated by both GLUT1 and GLUT4.