We have cloned the human UDP-N-acetylglucosamine (UDP-GlcNAc) transporter cDNA, which was recognized through a homology search in the expressed sequence tags database (dbEST) based on its similarity to the human UDP-galactose transporter. The chromosomal location of the UDP-GlcNAc transporter gene was assigned to chromosome 1p21 by fluorescence in situ hybridization (FISH). The transporter was expressed ubiquitously in every tissue so far examined. Expression of the transporter cDNA in CHO-Kl cells in its native and in a C-terminally HA-tagged form indicated that the human UDP-GleNAc transporter was localized in the Golgi apparatus. The membrane vesicles prepared from yeast cells expressing the cDNA product exhibited UDP-GleNAc-specific transporting activity. Comparison among UDP-galactose, CMP-sialic acid, and UDP-GIcNAc transporters from several organisms enabled us to identify residues highly conserved among the transporters and residues specific for each group of transporters.