Mechanistic studies have been carried out on the bacterial enzyme UDP-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This enzyme is interesting because it epimerizes a stereocenter that does not bear an acidic proton, and therefore it cannot utilize a simple deprotonation/reprotonation mechanism. A coupled enzyme assay employing UDP-ManNAc dehydrogenase has been developed. The epimerization in D2O is found to be accompanied by the incorporation of deuterium into the C-2 '' position of both epimers, supporting a mechanism that ultimately involves a proton transfer at this position. The epimerization of [2 ''-H-2]UDP-GlcNAc is slowed by a primary kinetic isotope effect indicating that C-H bond cleavage is occurring during a rate-determining step of the reaction. A positional isotope exchange (PM) experiment shows that an O-18 label in the sugar-UDP bridging position will scramble into nonbridging diphosphate positions during enzymatic epimerization. These observations are consistent with a mechanism that proceeds via cleavage of the anomeric C-O bond, with 2-acetamidoglucal and UDP as enzyme-bound intermediates. Additional evidence for this mechanism is found in the unusual observation that during extended incubations, the intermediates are gradually released from the enzyme and accumulate in solution. These intermediates are formed by an anti elimination of UDP from UDP-GlcNAc and a syn elimination of UDP from UDP-ManNAc. It is likely that El-like eliminations via oxocarbenium intermediates are involved in the reaction. Further experiments show that 3 ''-deoxy-UDP-GlcNAc is not a substrate for the enzyme and that the enzyme does not contain a tightly bound NAD(+) cofactor.