Four genes, ORF 22 approximately 25, from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22 were analyzed for their involvement in the biosynthesis of the two deoxysugar moieties of the granaticins. Each gene was individually inactivated on a cosmid carrying the entire gra gene cluster and the mutant cosmids were transformed into S. coelicolor CH999. Analysis of the pattern of pigment production by the transformants revealed that each of the four ORFs is required for the formation/attachment of the L-rhodinose moiety of granaticin B, but not that of the D-olivose moiety of granaticin. Based on these results and sequence homologies a pathway of dTDP-L-rhodinose formation is proposed which implicates ORF23, and possibly also ORF 24, in the 3-deoxygenation reaction, ORF 25 in the epimerization and ORF 22 in the final 4-ketoreduction.