In Salmonella typhimurium, three groups of genes located in rfb, rfe, and rff clusters are known to be involved in the biosynthesis of the enterobacterial common antigen. We found that enzymatic synthesis of uridine diphosphate N-acetylmannosaminouric acid, the activated form of a constituent sugar of the common antigen, followed the pathway previously described in Escherichia coli (N. Ichihara, N. Ishimoto, and E. Ito, FEBS Lett. 39:46--48, 1974). All of the six rff mutants tested, which fail to synthesize the common antigen, were deficient in one or both of the two enzymes needed for the synthesis of this sugar nucleotide from uridine diphosphate N-acetylglucosamine; these results established the physiological role of the pathway studied for the biosynthesis of N-acetylmannosaminuronic acid residues. The levels of these enzymes were not reduced in rfe mutants or rfb deletion mutants, although they produced no or only traces of the common antigen.