<jats:p>The polyoxin (POL) biosynthetic gene cluster (<jats:italic>pol</jats:italic>) was recently cloned from <jats:italic>Streptomyces cacaoi</jats:italic> subsp. <jats:italic>asoensis</jats:italic>. A 3.3 kb DNA fragment carrying an obvious open reading frame (<jats:italic>polR</jats:italic>), whose deduced product shows sequence similarity to SanG of <jats:italic>Streptomyces ansochromogenes</jats:italic> and PimR of <jats:italic>Streptomyces natalensis</jats:italic>, was revealed within the <jats:italic>pol</jats:italic> gene cluster. Disruption of <jats:italic>polR</jats:italic> abolished POL production, which could be complemented by the integration of a single copy of <jats:italic>polR</jats:italic> into the chromosome of the non-producing mutant. The introduction of an extra copy of <jats:italic>polR</jats:italic> in the wild-type strain resulted in increased production of POLs. The transcription start point (tsp) of <jats:italic>polR</jats:italic> was determined by S1 mapping. Reverse transcriptase PCR experiments showed that PolR is required for the transcription of 18 structural genes in the <jats:italic>pol</jats:italic> gene cluster. Furthermore, we showed that <jats:italic>polC</jats:italic> and <jats:italic>polB</jats:italic>, the respective first genes of two putative operons (<jats:italic>polC–polQ2</jats:italic> and <jats:italic>polA–polB</jats:italic>) consisting of 16 and 2 of these 18 genes, have similar promoter structures. Gel retardation assays indicated that PolR has specific DNA-binding activity for the promoter regions of <jats:italic>polC</jats:italic> and <jats:italic>polB</jats:italic>. Our data suggest that PolR acts in a positive manner to regulate POL production by activating the transcription of at least two putative operons in the <jats:italic>pol</jats:italic> gene cluster.