Glucose deprivation dramatically increases glucose transport activity in 3T3-L1 adipocytes without changing the concentration of GLUT1 in the plasma membrane. Recent data suggest that subcompartments within the plasma membrane, specifically lipid rafts, may sequester selected proteins and alter their activity. To evaluate this possibility, we have examined the distribution of GLUT1 in Triton X-100 soluble and insoluble fractions. Our data show that 77% of the GLUT1 pool in plasma membranes isolated from control 3T3-L1 adipocytes was extracted by 0.2% Triton X-100. After glucose deprivation for 12h, only 56% of GLUT1 was extracted by detergent. In contrast, there was a 2.0-fold increase in the GLUT1 content of the detergent-resistant fraction. To evaluate whether GLUT1 interacts with a specific protein within lipid rafts, we focused on stomatin, which was recently shown to interact with and inhibit GLUT1 activity. Stomatin is distributed about equally between the plasma membrane and the biosynthetic compartments and its expression is not affected by glucose deprivation. Nearly 90% of the plasma membrane pool of stomatin is in detergent-resistant lipid rafts. In normal 3T3-L1 adipocytes, we were unable to demonstrate an interaction between GLUT1 and stomatin in co-immunoprecipitation experiments. However, in stomatin-overexpressing cells, there was clear co-precipitation of stomatin with GLUT1 antibodies. Glucose deprivation increased this interaction by 3-fold which may reflect the increase of GLUT1 in lipid rafts. Despite this, there was little change in transport activity in glucose-deprived, stomatinoverexpressing cells versus that in control cells. Thus, GLUT1 interacts with stomatin in lipid rafts, but this interaction per se does not alter transport activity. Rather stomatin may serve as an anchor for GLUT1 in lipid rafts, the environment of which favors activation.