Comamonas testosteroni TA441 adapts to utilization of phenol upon incubation with phenol as the major carbon source. Strain TA441 has a cluster of genes (aphKLMNOPQB) encoding the catabolic enzymes phenol hydroxylase and catechol 2,3-dioxygenase, and a divergently transcribed regulatory gene (aphR), but these genes are silent until adaptation occurs. We found another regulatory gene (aphS) downstream of aphR. AphS belongs to the GntR family of transcriptional regulators. All adapted strains were found to have mutations in the aphS gene or in the aphR-aphS intervening region. The adapted strains expressed phenol hydroxylase and catechol 2, 3-dioxygenase activity in the presence of phenol. The transcriptional activity of both the aphK and the aphR promoters was elevated in the adapted strains. A strain whose aphS gene was artificially disrupted was found to be able to grow using phenol, and the cells showed high levels of the above-mentioned transcriptional and enzymatic activities, indicating that adaptation was caused only by the mutation in the aphS gene. Gel retardation analysis revealed that AphS bound to two specific sites in the promoter region between aphK and aphR. These results indicate that the active aphS gene product acts as a trans-acting factor and represses transcription of the aph genes in strain TA441.