<jats:p> In a survey of microbial systems capable of generating unusual metabolite structural variability, <jats:italic>Streptomyces venezuelae</jats:italic> ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus ( <jats:italic>pikA</jats:italic> ) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis ( <jats:italic>des</jats:italic> ) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by <jats:italic>desVII</jats:italic> ) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the <jats:italic>pikC</jats:italic> -encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the <jats:italic>pik</jats:italic> gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.