Ossamycin from Streptomyces hygroscopicus var. ossamyceticus is an antifungal and cytotoxic polyketide and a potent inhibitor of the mitochondrial ATPase. Analysis of a near-complete genome sequence of the ossamycin producer has allowed the identification of the 127-kbp ossamycin biosynthetic gene cluster. The presence in the cluster of a specific crotonyl-CoA carboxylase/reductase homologue suggests that the 5-methylhexanoate extension unit used in construction of the macrocyclic core is incorporated intact from the unusual precursor isobutyrylmalonyl-CoA. Surprisingly, the modular polyketide synthase uses only 14 extension modules to accomplish 15 cycles of polyketide chain extension, a rare example of programmed iteration on a modular polyketide synthase. Specific deletion of genes encoding cytochrome P450 enzymes has given insight into the late-stage tailoring of the ossamycin macrocycle required for the attachment of the unusual 2,3,4,6-deoxyaminohexose sugar l-ossamine to C-8 of the ossamycin macrocycle. The ossamycin cluster also encodes a putative spirocyclase enzyme, OssO, which may play a role in establishing the characteristic spiroketal moiety of the natural product.