Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.