Aims: Ste15 and ste22 present in the Ebosin biosynthesis gene cluster (ste) were previously shown to function in Ebosin biosynthesis and both of the protein products are predicted to be glycosyltransferases. In this study, their biochemical activities were confirmed. Methods and Results: ste15 and ste22 were cloned and expressed in Escherichia coli. With a continuous coupled spectrophotometric assay and using the purified proteins, we now demonstrated that the protein Ste15 has the ability of catalysing the transfer of glucose specifically from UDP-glucose to an Ebosin precursor that lacks glucose, the lipid carrier located in the cytoplasmic membrane of the gene ste15 disrupt mutant Streptomyces sp. 139 (ste15-). The protein Ste22 can catalyse the transfer of rhamnose specifically from TDP-rhamnose to an Ebosin precursor that lacks rhamnose, a lipophilic carrier in the cytoplasmic membrane of the gene ste22 disrupt mutant Streptomyces sp. 139 (ste22-). Conclusions: The gene product of ste15 was identified to be a glucosyltransferase, and the protein encoded by ste22 was found to be a rhamnosyltransferase. Significance and Impact of the Study: Both of two enzymes play essential roles in the formation of repeating units of sugars during Ebosin biosynthesis. These are the first glucosyltransferase and rhamnosyltransferase in the biosynthesis of a Streptomyces exopolysaccharide to be characterized.