<jats:p>DNA from a region downstream of and overlapping the polyketide synthase (PKS) gene cluster for jadomycin B biosynthesis in <jats:italic>Streptomyces venezuelae</jats:italic> was cloned and sequenced. Analysis of the nucleotide sequence located one complete ORF (ORF6), an incomplete one representing the 3' region of ORF4 in the PKS cluster, and a second incomplete one (ORF7). The deduced amino acid sequences for ORFs 6 and 7 resemble those of oxygenases. Since a plausible biosynthetic pathway for jadomycin B includes an angular polyketide intermediate that undergoes oxidative ring fission before condensation with an amino acid, we subcloned one of the presumptive oxygenase genes (ORF6) in a segregationally unstable shuttle vector (pHJL400) and disrupted it by inserting the gene for apramycin resistance. Transformation of <jats:italic>S. venezuelae</jats:italic> with the disruption vector and selection for apramycin resistance gave mutants blocked in jadomycin biosynthesis. Southern hybridization confirmed that gene replacement had occurred. Cultures of the mutants accumulated a metabolite identified by comparison with an authentic sample as rabelomycin, a non-nitrogenous polyketide-derived antibiotic originally isolated from <jats:italic>Streptomyces olivaceus.</jats:italic>