Sequencing of the eryA region of the erythromycin biosynthetic gene cluster from Saccharopolyspora erythraea has revealed another structural gene (ORF B), in addition to the previously characterised ORF A, which appears to encode a component of 6-deoxyerythronolide-B synthase, the enzyme that catalyses the first stage in the biosynthesis of the polyketide antibiotic erythromycin A. The nucleotide sequence of ORF B, which lies immediately adjacent to ORF A, has been determined. The predicted gene product of ORF B is a polypeptide of 374417 Da (3568 amino acids), which is highly similar to the product of ORF A and which likewise contains a number of separate domains, each with substantial amino acid sequence similarity to components of known fatty-acid synthases and polyketide synthases. The order of the predicted active sites along the chain from the N-terminus is 3-oxoacyl-synthase--acyltransferase--acyl-carrier-protein-- 3-oxoacyl-synthase--acyltransferase--dehydratase--enoylreductase-- oxoreductase--acyl-carrier-protein. The position of the dehydratase active site has been pinpointed for the first time for any polyketide synthase or vertebrate fatty-acid synthase. The predicted domain structure of 6-deoxyerythronolide-B synthase is strikingly similar to that previously established for vertebrate fatty-acid synthases. This analysis of the sequence supports the view that the erythromycin-producing polyketide synthase contains three multienzyme polypeptides, each of which accomplishes two successive cycles of polyketide chain extension. In this scheme, the role of the ORF B gene product is to accomplish extension cycles 3 and 4.