THE role of G proteins in glucose uptake was investigated using C6 glioma cells. Carbachol (an agonist acting via G protein coupled receptors) and 5′-guanylylimido diphosphate (Gpp(NH)p; a nonhydrolysable guanine nucleotide analog which bypasses the receptors and directly activates G proteins) stimulated [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells, suggesting that hexose uptake is a G protein-mediated process. To identify the G protein involved in glucose uptake by C6 cells, the effect of carbachol on 2DG uptake was examined in the presence of pertussis toxin. Pertussis toxin treatment did not alter the ability of C6 cells to respond to carbachol, ruling out the involvement of Giαin 2DG uptake. C6 cells were transfected with Gqαor GLUT1 cDNA for 48 h, exposed to 1 mM carbachol for 2 h, and processed for 2DG uptake. Carbachol stimulated 2DG uptake in both Gqαand GLUT1-transfected cells. Gpp(NH)p, also stimulated 2DG uptake in Gqαand GLUT1-transfected cells. These results suggest that muscarinic receptor coupling to Gqαregulates hexose uptake in C6 cells.