Genes involved in deoxysugar metabolism, encoding thymidine diphospho (TDP)-glucose 4,6-dehydratase (gdh) and a putative TDP-4-keto-6-deoxyglucose 3,5-epimerase (kde), were cloned from the erythromycin (Er)-producing Saccharopolyspora erythraea by means of an oligodeoxynucleotide corresponding to a segment of the purified Gdh protein. Determination of the nucleotide sequence established that kde lies 3' to gdh. The function of gdh was confirmed by an enzymatic assay following expression of the gene in Escherichia coli. Southern analysis indicated that Sa. erythraea contains only one copy of gdh and kde. It was not possible to establish whether these genes are required for Er biosynthesis, but they appear to be essential for cellular metabolism, since resolution of a partial diploid containing a wt and a disrupted copy of gdh always maintained the wt gene. These loci do not lie within or near the known boundaries of the cluster of Er-production and -resistance genes, nor do they appear to be flanked by other deoxysugar biosynthesis genes.