When MDCK cells were incubated in the presence of the protein synthesis inhibitor puromycin or cycloheximide, there was a rapid and concentration-dependent inhibition in the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and into protein. However, mannose incorporation into dolichyl-P-mannose was not affected. Interestingly, these inhibitors did block [6-3H]glucosamine incorporation into dolichyl-PP-GlcNAc as well as into lipid-linked oligosaccharides. Similar results were obtained when other cell lines were used and also when inhibitors of protein glycosylation such as beta-hydroxynorvaline and beta-fluoroasparagine were used. Cells incubated in puromycin did not show any changes in the levels of sugar nucleotides, GDP-mannose or UDP-GlcNAc, or in the in vitro activities of the glycosyltransferases that add mannose to the lipid-linked oligosaccharides. The inhibition of mannose incorporation into lipid-linked oligosaccharides could not be overcome by addition of dolichyl-P to the inhibited cells, even though the addition of dolichyl-P to control cells stimulated mannose incorporation into dolichyl-P-mannose, lipid-linked oligosaccharides, and protein from 3- to 5-fold. Thus, limitations in the levels of dolichyl-P do not appear to be a major factor in this inhibition. On the other hand, addition of the tripeptide acceptor N-acyl-Asn-Try-Thr did overcome the puromycin inhibition to some extent, suggesting that accumulation of some intermediate such as lipid-linked oligosaccharides might be involved in the inhibition.