Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at -10 degrees C for 1 month. The solubilized enzyme synthesized [(14)C]Man(3)GlcNAc(2)-pyrophosphoryl-polyprenol and [(14)C]Man(5)GlcNAc(2)-pyrophosphoryl-polyprenol when incubated with GDP-[(14)C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid-linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man(3)GlcNAc(2)-lipid and Man(5)GlcNAc(2)-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man(6)GlcNAc(2)-lipid, Man(7)GlcNAc(2)-lipid, and Man(8)GlcNAc(2)-lipid occurred when the above incubations were supplemented with dolichyl-phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form Man(5)GlcNAc(2)-lipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.