Upon incubation of UDP-N-acetyl[14C]glucosamine and GDP-mannose with rough microsomes from rat liver, GlcNAc incorporation occurred mostly into acid-labile products extractable by chloroform/methanol that were presumed to be dolichol pyrophosphate GlcNAc and, for a small part, dolichol pyrophosphate N,N'-diacetylchitobiose. The production of this latter glycolipid was greatly enhanced when rough microsomes, previously treated with pyrophosphate, were incubated in the presence of GTP. Furthermore, under these particular conditions, high levels of radioactivity were obtained in products precipitated by trichloroacetic acid and not extracted by chloroform/methanol. The label appeared in acid-labile and in acid-resistant products, presumed to be dolichol pyrophosphate mannosylated oligosaccharides, and glycoproteins, respectively. From these results, dolichol pyrophosphate N,N'-diacetylchitobiose appears to be an obligate metabolic intermediate in protein glycosylation by this system. Whether or not dolichol pyrophosphate N,N'-diacetylchitobiose must be mannosylated prior to transfer of the N,N'-diacetylchitobiose moiety to protein is an open matter, because GlcNAc was also incorporated into acid-resistant products when the reaction was carried out in absence of GDP-mannose.