Previous studies suggest that regulation of hexose uptake in Chinese hamster ovary fibroblasts can occur by alterations in glucose transporter intrinsic activity without changes in cell surface transporter number (Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., and Czech, M. P. (1990) J. Biol. Chem. 265, 5793-5801). We tested this hypothesis using 3T3-L1 fibroblasts and adipocytes which exhibit 5-6-fold increases in 2-deoxyglucose or 3-O-methylglucose uptake when exposed to low micromolar concentrations of cadmium for 18 h. Cadmium treatment decreased the apparent Km of 3T3-L1 fibroblasts for 3-O-methylglucose influx from approximately 28 to 9 mM and increased the apparent Vmax by 2-3-fold. These fibroblasts lack the skeletal muscle/adipocyte-type (GLUT4) transporter and showed only a small increase in total cellular immunoreactive HepG2 type (GLUT1) transporter in response to cadmium. Furthermore, cell surface GLUT1 levels did not change in 3T3-L1 fibroblasts exposed to cadmium, as assessed by the binding to intact cells of an antibody which recognizes an extracellular GLUT1 epitope. Insulin enhanced 2-deoxyglucose uptake 2-fold in 3T3-L1 fibroblasts, but did not further stimulate cadmium-activated transport rates. In contrast, insulin stimulated hexose transport 15-fold in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4 proteins, and this effect was fully additive with the 5-fold effect of cadmium. Cadmium had little or no effect on immunoreactive GLUT1 or GLUT4 in isolated 3T3-L1 adipocyte plasma membranes. In contrast, insulin action led to marked recruitment (3-fold) of GLUT4 to the plasma membrane fraction in adipocytes treated with or without cadmium. Taken together, these data are consistent with the hypothesis that cadmium-activated sugar uptake is catalyzed by GLUT1, whereas insulin-stimulated sugar uptake is catalyzed predominantly by GLUT4 in 3T3-L1 adipocytes. Furthermore, the data suggest that the GLUT1 transporter can undergo significant increases in intrinsic catalytic activity in response to cadmium treatment of 3T3-L1 fibroblasts and adipocytes.