The α- and β-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyl-transferase reactions. The α analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the βS analogs were approximately 10 times more resistant. The βS analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4β-galactosyltransferase exhibited a Vmax with (βS) UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4β-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the βS analog in each system.