The methymycin and pikromycin series of antibiotics are structurally related macrolides produced by several Streptomyces species, including Streptomyces venezuelae ATCC 15439, which produces both 12-membered ring macrolides methymycin, neomethymycin, and 14-membered ring macrolides pikromycin and narbomycin. Cloning and sequencing of the biosynthetic gene clusters for these macrolides from three selected Streptomyces strains revealed a common genetic architecture of their polyketide synthases (PKSs). Unlike PKS clusters of other 14-membered ring macrolides such as erythromycin and oleandomycin, each of the pikromycin series producers harbors a six module PKS cluster, in which modules 5 and 6 are encoded on two separate proteins instead of one bimodular protein, as well as a thioesterase II gene immediately downstream of the main PKS gene. The results shed new light on the evolution of modular PKSs and provide further evidence on the regulation of methymycin and pikromycin production in S. venezuelae ATCC 15439.