Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates ( j . Biol. Chem. 261 :16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L, myocytes at 16 m M glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 p,M) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 p, M, cyclohexirnide prevented the rnodulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicarnycin A , reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinornycin D prevented the downregulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steadystate dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.