Many studies suggest that sulfonylureas (SUs) have direct extrapancreatic actions. The action of gliclazide, a new SU, was examined and compared to that of glyburide in L6 myotubes, a model of skeletal muscle. Gliclazide and glyburide increased 2-deoxy-D-glucose (2DG) uptake in a time- and dose-dependent fashion after 24 h to a maximum of 179% and 202% of the basal value, respectively (P < 0.001). Acute (30-min) insulin (10(-7) M) stimulated 2DG uptake to similar levels (203% of basal), but this effect was absent after maximum stimulation by SU. SU action did not require insulin and was not blocked by the protein synthesis inhibitor cycloheximide. To investigate the mechanism of stimulation of 2DG uptake, cells were fractionated, and total plasma membrane and internal membrane levels of glucose transporter (GLUT) isoforms were determined by immunoblotting. Both drugs significantly increased the total content (1.7-fold) and plasma membrane level (1.8-fold) of GLUT1, with no change in internal membrane. Total content and plasma membrane levels of GLUT4 and GLUT3 did not change or showed a small decrease. We conclude that the stimulation of glucose uptake in L6 cells by gliclazide and glyburide is associated not with a redistribution but, rather, with an increase in the total membrane content and plasma membrane level of GLUT1, which is independent of protein synthesis. These data suggest a novel action of SU to stabilize GLUT1 protein at the plasma membrane.