Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal alpha1-->2-linked N-acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins. We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent Km of 5.5 microM and a Vmax of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids.