A 10.2-kb fragment of DNA from Streptomyces roseofulvus, which contains polyketide synthase (PKS)-encoding genes (fren) presumed to determine production of the antibiotics frenolicin and the nanaomycins, was cloned. A 5530-bp continuous segment of this DNA was sequenced. Analysis of the sequence revealed five complete open reading frames (ORFs) transcribed in one direction (ORFs 1, 2, 3, 5, 4) and one (ORFX), located between ORF3 and ORF5, transcribed in the opposite direction. The deduced amino-acid sequences of ORFs 1, 2, 3, 4 and 5 closely resemble the sequences of known components of the type-II PKS from other Streptomyces species: putative heterodimeric (ORF1 + 2) ketosynthase, acyl carrier protein, cyclase and ketoreductase, respectively. A resemblance between the N-terminal and C-terminal halves of the ORF4 product--also discovered in the corresponding genes from other isochromanequinone antibiotic producers--suggests a possible origin of the cyclase-encoding gene by duplication. ORFX appears to represent a novel class of genes of unknown function present not only in the fren cluster, but also in other clusters of aromatic antibiotic biosynthetic genes in Streptomyces species. The fren-ORF1-5 genes, encoding a PKS that constructs a nascent polyketide of either 16 or 18 carbons, compared with fixed lengths of 16 and 20 for other available examples, are proving to be valuable for understanding the mechanisms controlling polyketide chain length and patterns of reduction and cyclisation.