NovU and novS represent C-methyltransferase and dTDP-4-keto-reductase putatively involved in noviose biosynthetic pathway. We have expressed these genes in E. coli and identified the functions of their corresponding products by in vitro enzyme assay. The results indicated that novU encodes a C-methyltransferase and catalyzes the methylation of the NovW product (dTDP-glucose 3,5-epimerase). Similarly, NovS encodes a dTDP-4-ketoreductase and catalyzes the formation of rhamnose utilizing dTDP-4-keto-6-deoxyglucose in the presence of NADH and NovW. Furthermore, the pathway for the biosynthesis of noviose has been confirmed.