The oviedomycin biosynthetic gene cluster from Streptomyces antibioticus ATCC 11891 has been sequenced and characterized. It contains all the necessary genes for oviedomycin biosynthesis, together with several genes for the generation of malonyl-CoA extender units. Production of this unusual angucyclinone in its natural host occurs only in solid cultures in parallel with aerial mycelium and spore formation. A mutant that did not produce oviedomycin was generated by disruption of the β-ketoacyl synthase gene ovmK. No other physiological process in the mutant appears to be affected; this rules out a direct relationship between oviedomycin production and cell differentiation in S. antibioticus.