Streptomyces cinnamonensis DSM 1042 produces two classes of secondary metabolites of mixed isoprenoid/nonisoprenoid origin: the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and several prenylated phenazines, predominantly endophenazine A. We now report the cloning and sequence analysis of a 55 kb gene cluster required for the biosynthesis of these compounds. Several inactivation experiments confirmed the involvement of this gene cluster in the biosynthesis of FNQ I and endophenazine A. The six identified genes for endophenazine biosynthesis showed close similarity to phenazine biosynthetic genes from Pseudomonas. Of the 28 open reading frames identified in the adjacent FNQ I cluster, 13 showed close similarity to genes contained in the cluster for furaquinocin-a structurally similar metabolite from another Streptomyces strain. These genes included a type III polyketide synthase sequence, a momA-like monooxygenase gene, and two cloQ-like prenyltransferase genes designated fnq26 and fnq28. Inactivation experiments confirmed the involvement of fnq26 in FNQ I biosynthesis, whereas no change in secondary-metabolite formation was observed after fnq28 inactivation. The FNQ I cluster contains a contiguous group of five genes, which together encode all the enzymatic functions required for the recycling of S-adenosylhomocysteine (SAH) to S-adenosylmethionine (SAM). Two SAM-dependent methyltransferases are encoded within the cluster. Inactivation experiments showed that fnq9 is responsible for the 7-O-methylation and fnq27 for the 6-C-methylation reaction in FNQ I biosynthesis.