<jats:title>Abstract</jats:title><jats:p>Griseoviridin (GV) and viridogrisein (VG, also referred to as etamycin), produced by <jats:italic>Streptomyces griseoviridis</jats:italic>, are two chemically unrelated compounds belonging to the streptogramin family. Both of these natural products demonstrate broad‐spectrum antibacterial activity and constitute excellent candidates for future drug development. To elucidate the biosynthetic machinery associated with production of these two unique antibiotics, the gene cluster responsible for both GV and VG production was identified within the <jats:italic>Streptomyces griseoviridis</jats:italic> genome and characterized, and its function in GV and VG biosynthesis was confirmed by inactivation of 30 genes and complementation experiments. This <jats:italic>sgv</jats:italic> gene cluster is localized to a 105 kb DNA region that consists of 36 open reading frames (ORFs), including four nonribosomal peptide synthetases (NRPSs) for VG biosynthesis and a set of hybrid polyketide synthases (PKS)‐NRPSs with a discrete acyltransferase (AT), SgvQ, to assemble the GV backbone. The enzyme encoding genes for VG versus GV biosynthesis are separated into distinct “halves” of the cluster. A series of four genes: <jats:italic>sgvA</jats:italic>, <jats:italic>sgvB</jats:italic>, <jats:italic>sgvC</jats:italic>, and <jats:italic>sgvK</jats:italic>, were found downstream of the PKS‐NRPS; these likely code for construction of a γ‐butyrolactone (GBL)‐like molecule. GBLs and the corresponding GBL receptor systems are the highest ranked regulators that are able to coordinate the two streptomyces antibiotic regulatory protein (SARP) family positive regulators SgvR2 and SgvR3; both are key biosynthetic activators. Models of GV, VG, and GBL biosynthesis were proposed by using functional gene assignments, determined on the basis of bioinformatics analysis and further supported by in vivo gene inactivation experiments. Overall, this work provides new insights into the biosyntheses of the GV and VG streptogramins that are potentially applicable to a host of combinatorial biosynthetic scenarios.