<jats:title>Summary</jats:title><jats:p>Genes for biosynthesis of a <jats:italic>Streptomyces</jats:italic> sp. FR‐008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in <jats:italic>Escherichia coli</jats:italic> using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthiesis by gene replacement. The FR‐008 antibiotic contains a 38‐membered, poiyketide‐derived macrolide ring. Southern hybridization using probes encoding domains of the type i modular erythromycin polyketide synthase (PKS) showed that the <jats:italic>Streptomyces</jats:italic> sp. FR‐008 PKS gene cluster contains repeated sequences spanning <jats:italic>c.</jats:italic> 105 kb of contiguous DNA; assuming <jats:italic>c.</jats:italic> 5 kb for each PKS module, this is in striking agreement with the expectation for the 21‐step condensation process required for synthesis of the FR‐008 carbon chain. The methods developed for transformation and gene replacement in <jats:italic>Streptomyces</jats:italic> sp. FR‐008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.