Lipid-linked oligosaccharides containing at least five mannose residues (0.7 nmol/g of tissue) were isolated from calf pancreas by chloroform-methanol-water extraction and purification on DEAE-cellulose and Sephadex LH-20, and affinity chromatography on concanavalin A-Sepharose in the presence of nonionic detergent. The addition, prior to the chromatographic steps, of 14C-labeled lipid-linked oligosaccharides (synthesized in vitro by calf pancreas microsomes in the presence of GDP-D-[14C]mannose and UDP-D-[14C]glucose, respectively) as internal standards, indicated a final yield ranging from 38 to 50%. Analysis of the oligosaccharide residues by liquid chromatography of the lipid-free preparation, monitored by u.v. absorbance and radioactivity measurement of the tritiated compounds, indicated a heterogeneous mixture of oligosaccharides. Its components, ranging from Man5(GlcNAc)2 to Glc3Man9(GlcNAc)2, cochromatographed with the 14C-labeled derivatives from in vitro synthesis. Calf pancreas contains lipid intermediates bearing at least six mannose residues, such as Man9(GlcNAc)2-P-P-lipid, in almost equal or even higher amounts than Glc3Man9(GlcNAc)2-P-P-dolichol.