ABSTRACTTherol(designated forresorcinol) gene clusterrolRHMDis involved in resorcinol catabolism inCorynebacterium glutamicum, and RolR is the TetR-type regulator. In this study, we investigated how RolR regulated the transcription of therolgenes inC. glutamicum. The transcription start sites and promoters ofrolRandrolHMDwere identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that RolR negatively regulated the transcription ofrolHMDand of its own gene. Further, a 29-bp operatorrolOwas located at the intergenic region ofrolRandrolHMDand was identified as the sole binding site for RolR. It contained two overlapping inverted repeats and they were essential for RolR-binding. The binding of RolR torolOwas affected by resorcinol and hydroxyquinol, which are the starting compounds of resorcinol catabolic pathway. These two compounds were able to dissociate RolR-rolOcomplex, thus releasing RolR from the complex and derepressing the transcription ofrolgenes inC. glutamicum. It is proposed that the binding of RolR to its operatorrolOblocks the transcription ofrolHMDand of its own gene, thus negatively regulated resorcinol degradation inC. glutamicum.