Based on the rationale that internalized 2-deoxy-D-glucose (dGlc) is toxic to cells, glucose transport (GLUT) defective myoblast mutants have been isolated by their ability to grow in glucose-free medium containing dGlc. Recent studies revealed that the GLUT 1 transport process was activated when GLUT 3-GLUT 4-mutants were grown in glucose-free medium. It was therefore puzzling why these GLUT3-GLUT4-myoblasts could survive in the presence of dGlc during the mutant selection process. The present study revealed that GLUT 1 transport affinity in dGlc-grown cells was at least four folds lower than that in control cells. This loss of GLUT 1 transport activity was apparent only after exposure to the toxic sugar analogues for more than 10 hrs. This dGlc-mediated effect was not due to competitive inhibition by the residual dGlc carried over from growth medium, changes in glycolytic enzymes, nor accumulation of the negatively charged dGlc-6-PO4. In fact, GLUT 1 transcript level was elevated in dGlc-treated cells. Both immunoprecipitation and immunoblotting studies indicated that the size of the GLUT 1 transporter in dGlc-grown myoblasts was reduced from 52 kDa to that of the unglycosylated form (38 kDa). These findings suggest that growth in the presence of dGlc inhibits glycosylation of the GLUT 1 transporter, thus reducing its transport affinity. This inability of the GLUT 1 transporter to take up dGlc may therefore explain why GLUT 3-GLUT 4-mutants are able to grow in the presence of the toxic dGlc during the mutant selection procedure.