Previous studies from several laboratories have reported that inhibition of protein synthesis results in a concomitant reduction in synthesis of oligosaccharide-diphosphoryldolichol. We have investigated this phenomenon in LM cells grown in defined culture medium. The results of this study indicate that incubation of LM cell with cycloheximide at a concentration sufficient to totally arrest polypeptide synthesis, results in a rapid reduction in the synthesis of [3H]mannose-labeled or [3H]glucosamine-labeled oligosaccharide-lipid within 2 min. Cycloheximide treatment had only a slight inhibitory effect on synthesis of GDP-Man. Furthermore, during the first 5 min after the addition of cycloheximide to LM cells, when [3H]mannose incorporation into oligosaccharide-lipid was maximally reduced, the specific activity of the GDP-Man pool was identical to that observed in control cells. Labeling in vivo of the lipid-linked saccharide precursors of oligosaccharide-lipid revealed that cycloheximide had virtually no effect on the synthesis of Man-P-Dol, GlcNAc-PP-Dol, GlcNAc2-PP-Dol, and beta-Man-(GlcNAc)2-PP-Dol (Dol = dolichol). In agreement with this finding, results of experiments in vitro using microsomes prepared from LM cells indicate that cycloheximide did not directly inhibit the enzymes responsible for the synthesis of these lipid-linked saccharide precursors. Supplementation of mouse LM cells by preincubation for 1 h with dolichyl phosphate (5 micrograms/ml) resulted in a 300% stimulation of oligosaccharide-lipid synthesis when compared to non-supplemented cells. However, dolichyl phosphate supplementation of cycloheximide-treated cells failed to restore oligosaccharide-lipid synthesis to the level observed in control cells. In control cells pre-labeled oligosaccharide-lipid turned over rapidly and, as expected, cycloheximide addition to the chase medium significantly retarded this turnover process. However, preincubation of cells with exogenous dolichyl phosphate had little or no effect on the turnover of oligosaccharide-lipid in control or cycloheximide-treated cells. These findings argue against dolichyl phosphate deficiency as the primary cause of reduced oligosaccharide-lipid synthesis when protein synthesis is blocked. The implications of these results, and an alternative hypothesis to explain the effect of inhibition of protein synthesis on oligosaccharide-lipid synthesis based on elevation of intracellular GTP levels, are discussed.