The bph genes in Pseudomonas sp. KKS102, which are involved in the degradation of polychlorinated biphenyl/biphenyl, are induced in the presence of biphenyl. In this study our goal was to understand the regulatory mechanisms involved in the inducible expression. The bph genes (bphEGF(orf4)A1A2A3BCD(orf1)A4R) constitute an operon, and its expression is strongly dependent on the pE promoter located upstream of the bphE gene. A bphS gene, whose deduced amino acid sequence showed homology with the GntR family transcriptional repressors, was identified at the upstream region of the bphE gene. Disruption of the bphS gene resulted in constitutive expression of bph genes, suggesting that the bphS gene product negatively regulated the pE promoter. The gel retardation and DNase footprinting analyses demonstrated specific binding of BphS to the pE promoter region and identified four BphS binding sites that were located within and immediately downstream of the -10 box of the pE promoter. The four binding sites were functional in repression because their respective elimination resulted in derepression of the pE promoter. The binding of BphS was abolished in the presence of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, an intermediate compound in the biphenyl degradation pathway. We concluded that the negative regulator BphS plays a central role in the regulation of bph gene expression through its action at the pE promoter.