The role of glycosylation of the carrier in the transporting activity was investigated in Swiss 3T3 cells. Inhibition of protein glycosylation by tunicamycin resulted in the decrease of hexose uptake in a dose- and time-dependent manner without a cytotoxic effect. From kinetic analysis, a decrease in the number or availability of hexose carriers in the plasma membrane was suggested. This was in good correlation with the decrease in the amount of photoaffinity cytochalasin B binding in the plasma membrane by the treatment with tunicamycin. The rate of phorbol 12,13-dibutyrate-induced translocation of the hexose carrier from microsomal to plasma membrane was reduced in tunicamycin-treated cells, which may be correlated with the decrease in the number of the completely glycosylated carrier translocatable from the microsomal membrane. In both tunicamycin-treated and untreated cells, the stimulation of hexose transport by phorbol 12,13-dibutyrate was abolished by the removal of phorbol 12,13-dibutyrate, and upon its readdition the stimulation recovered to the same degree as before the removal. Thus, the recycling of the functionally mature hexose carrier appeared not to be affected by the treatment with tunicamycin. These results suggested that complete glycosylation of the carrier may be necessary for the translocation of the carrier from microsomal to plasma membrane to accomplish its function on the cell surface.