Glc3Man9GlcNAc2-P-P-dolichol and Man9GlcNAc2-P-P-dolichol isolated from Saccharomyces cerevisiae are substrates for the N-glycosylation of endogenous proteins in Triton X-100-solubilized yeast microsomes. The solubilized oligosaccharide transferase requires Mn2+ for activity; neither Mg2+ nor Ca2+ is an effective substitute. The pH optimum of the transfer reaction is between 6.5 and 7.5. Unlike animal systems, which utilize glucosylated oligosaccharide-lipid to a much greater extent than the unglucosylated species as a donor in the transferase rection, yeast extracts transfer more than 70% of Glc3Man9GlcNAc2 and Man9GlcNAc2 from their respective oligosaccharide-lipids to proteins. However, the rate of N-glycosylation in vitro is approximately 25-fold faster with Glc3Man9GlcNAc2-P-P-dolichol than with Man9GlcNAc2-P-P-dolichol. The apparent Km value for the glucosylated species is 75 nM, while that for the unglucosylated glycolipid is 55 nM.