A [3H]mannose suicide selection has been used to isolate mutants in yeast which contain temperature-sensitive defects in asparagine-linked glycosylation. The surviving cells were screened at the nonpermissive temperature for a decreased ability to incorporate [3H]mannose and for defects in glycosylation of the secreted protein invertase. One of these mutants (alg1-1) has been characterized and found to be blocked in the assembly of the lipid-linked oligosaccharide precursor. The alg1-1 cells synthesize mannosyl compounds at 60% of the wild type level at the nonpermissive temperature and 105% of the wild type level at the permissive temperature. In vivo labeling experiments have demonstrated that alg1-1 cells are able to synthesize GlcNAc2-lipid but are unable to synthesize any mannose-containing oligosaccharide-lipids. This result was confirmed by in vitro labeling of yeast membranes. When incubated with UDP-[3H]GlcNAc, alg1-1 membranes synthesized GlcNAc2-lipid but failed to elongate it when GDP-Man was added. The alg1-1 membranes also failed to elongate exogenous GlcNAc2-lipid but were able to convert Man1GlcNAc2-lipid to Man5-Glc-NAc2-lipid in the presence of GDP-Man. These results indicate that the alg1-1 mutant is blocked specifically in the addition of the first mannose residue to the lipid-linked oligosaccharide precursor.