A search of the yeast data base for a protein homologous to Escherichia coli UDP-N-acetylglucosamine pyrophosphorylase yielded UAP1 (UDP-N-acetylglucosamine pyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively. Sequence analysis revealed that the human UAP1 cDNA was identical to previously reported AGX1. A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when expressed under the control of ScUAP1 promoter, both C. albicans and Homo sapiens UAP1 (CaUAP1 and HsUAP1) rescued the ScUAP1-deficient S. cerevisiae cells. All the recombinant ScUap1p, CaUap1p, and HsUap1p possessed UDP-N-acetylglucosamine pyrophosphorylase activities in vitro. The yeast Uap1p utilized N-acetylglucosamine-1-phosphate as the substrate, and together with Agm1p, it produced UDP-N-acetylglucosamine from N-acetylglucosamine-6-phosphate. These results demonstrate that the UAP1 genes indeed specify eukaryotic UDP-GlcNAc pyrophosphorylase and that phosphomutase reaction precedes uridyltransfer. Sequence comparison with other UDP-sugar pyrophosphorylases revealed that amino acid residues, Gly112, Gly114, Thr115, Arg116, Pro122, and Lys123 of ScUap1p are highly conserved in UDP-sugar pyrophosphorylases reported to date. Among these amino acids, alanine substitution for Gly112, Arg116, or Lys123 severely diminished the activity, suggesting that Gly112, Arg116, or Lys123 are possible catalytic residues of the enzyme.