Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.