The serotype c-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans NCTC 9710 contains an unusual sugar, 6-deoxy-L-talose, which has been identified as a constituent of cell wall components in some bacteria. Two genes coding for thymidine diphosphate (dTDP)-6-deoxy-L-lyxo-4-hexulose reductases were identified in the gene cluster required for biosynthesis of serotype c-specific polysaccharide. Both dTDP-6-deoxy-L-lyxo-4-hexulose reductases were overproduced and purified from Escherichia coli transformed with the plasmids containing these genes. The sugar nucleotides converted by both reductases were purified by reversed-phase high performance liquid chromatography and identified by (1)H nuclear magnetic resonance and gas-liquid chromatography. The results indicated that one of two reductases produced dTDP-6-deoxy-L-talose and the other produced dTDP-L-rhamnose (dTDP-6-deoxy-L-mannose). The amino acid sequence of the dTDP-6-deoxy-L-lyxo-4-hexulose reductase forming dTDP-6-deoxy-L-talose shared only weak homology with that forming dTDP-L-rhamnose, despite the fact that these two enzymes catalyze the reduction of the same substrate and the products are determined by the stereospecificity of the reductase activity. Neither the gene for dTDP-6-deoxy-L-talose biosynthesis nor its corresponding protein product has been found in other bacteria; this biosynthetic pathway is identified here for the first time.