The effect of tunicamycin, an inhibitor of protein glycosylation, on the high-affinity transport of D-aspartate was investigated in C6 astrocytoma cells. A concentration of tunicamycin (1 microgram/ml) that after 24 h exposure inhibited the rate of transport by 70% and incorporation of [3H]mannose by 82-95% had only a small effect on [14C]leucine incorporation into protein and cell growth (20% reduction). Tunicamycin decreased the Vmax for transport without affecting the Km, which suggests that inhibition of glycosylation reduces the number of competent transporters on the surface of the plasma membrane. The decrease in the velocity of uptake was attenuated when C6 cells were treated with tunicamycin in the presence of protease inhibitors, indicating that the underglycosylated carriers are subject to enhanced proteolytic degradation. Incubation in drug-free medium following treatment with 1 microgram/ml of tunicamycin for 24 h resulted in recovery of D-aspartate transport within 48 h. This recovery was prevented by the presence of cycloheximide, which indicates that synthesis of new transporters is necessary for the restoration of normal rates of D-aspartate uptake. These results support our earlier postulate that the high-affinity carriers for amino acid transmitters are transmembrane glycoproteins.