A series of synthetic nucleotide sugars have been studied as monosaccharide donors in the biosynthetic assembly of the O-antigen repeating units of Salmonella sero-groups E, B, C2 and C3. UDP-Tal did not serve as a substrate of galactosyl phosphate transferase, and GDP-Tal did not substitute GDP-Man in mannosyltransferase reactions. dTDP-L-Man proved to be a substrate of rhamnosyl transferases; GDP-2dMan, GDP-3dMan, GDP-D-Rha and GDP-Glc served as substrates for mannosyltransferases. The modified tri- and tetrasaccharide derivatives can be converted into the modified O-specific polysaccharides after incubation with Salmonella membranes.