We identified an open reading frame, designated phcS, downstream of the transcriptional activator gene (phcR) for the expression of multicomponent phenol hydroxylase (mPH) in Comamonas testosteroni R5. The deduced product of phcS was homologous to AphS of C. testosteroni TA441, which belongs to the GntR family of transcriptional regulators. The transformation of Pseudomonas aeruginosa PAO1c (phenol negative, catechol positive) with pROR502 containing phcR and the mPH genes conferred the ability to grow on phenol, while transformation with pROR504 containing phcS, phcR, and mPH genes did not confer this ability. The disruption of phcS in strain R5 had no effect on its phenol-oxygenating activity in a chemostat culture with phenol. The phenol-oxygenating activity was not expressed in strain R5 grown in a chemostat with acetate. In contrast, the phenol-oxygenating activity in the strain with a knockout phcS gene when grown in a chemostat with acetate as the limiting growth factor was 66% of that obtained in phenol-grown cells of the strain with a knockout in the phcS gene. The disruption of phcS and/or phcR and the complementation in trans of these defects confirm that PhcS is a trans-acting repressor and that the unfavorable expression of mPH in the phcS knockout cells grown on acetate requires PhcR. These results show that the PhcS protein repressed the gratuitous expression of phenol-metabolizing enzymes in the absence of the genuine substrate and that strain R5 acted by an unknown mechanism in which the PhcS-mediated repression was overcome in the presence of the pathway substrate.