A new membrane preparation from Saccharomyces cerevisiae was developed, which effectively catalyzes the synthesis of large oligosaccharide-lipids from GDP-Man and UDP-Glc allowing a detailed study of their formation and size. The oligosaccharide from an incubation with GDP-Man could be separated by gel filtration chromatography into several species consisting of two N-acetylglucosamine (GlcNAc) residues at the reducing end and differing by one mannos unit; the major compound formed has the composition (Man)9(GlcNAc)2. Upon incubation with UDP-Glc, three oligosaccharides corresponding to the size of (Glc)1-3(Man)9(GlcNAc)2 are formed. Thus, the oligosaccharides generated in vitro by the yeast membranes appear to be identical in size with the oligosaccharides found in animal systems. In addition the results indicate that dolichyl phosphate mannoe (DolP-Man) is the immediate donor in assembling the oligosaccharide moiety from (Man)5(GlcNAc)2 to (Man)9(GlcNAc)2. All three glucose residues are transferred from DolP-Glc. Experiments with isolated [Glc-14C]oligosaccharide-lipid as substrate demonstrated that the oligosaccharide chain is transferred to an endogenous membrane protein acceptor. Moreover, transfer is followed by an enzymic removal of glucose residues, due to a glucosidase activity associated with the membranes. Glucose release from the free [Glc-14C]oligosaccharide is less effective than from protein-bound oligosaccharide. Glycosylation was also observed using [Man-14C]oligosaccharide-lipid or DolPP-(GlcNAc)2 as donor. However, transfer in the presence of glucose seems to be more rapid. The mannose-containing oligosaccharide, released from the lipid, was shown to function as a substrate for further chain elongation reactions utilizing GDP-Man but not DolPP-Man as donor. It is suggested that the immediate precursor in the synthesis of the heterogeneous core region, (Man)12-17(GlcNAc)2, of yeast mannoproteins is a glucose-containing lipid-oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2, i.e. only part of what has been defined as inner core is built up on the lipid carrier. After transfer to protein the oligosaccharide is modified by excision of the glucose residues, followed subsequently by further elongation from GDP-Man to give the size of th oligosaccharide chains found in native mannoproteins.