We have used site-directed mutagenesis to analyze the importance of nucleotides in the catBC promoter region for the binding of CatR, a member of the LysR family of DNA-binding proteins and for the in vivo activation of the catBC operon. The binding affinity of CatR for its binding site in the catBC promoter region was shown to increase about 2.2-fold in the presence of the inducer, cis,cis-muconate. Nucleotides were targeted for mutagenesis on the basis of previous footprinting data and sequence analysis of CatR binding sites. Critical nucleotides for CatR binding were found within an imperfect inverted repeat. Previous studies have indicated that the LysR family of DNA-binding proteins shares a consensus T-N11-A binding motif (Goethals, K., Van Montagu, M., and Holsters, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1646-1650), but the CatR binding site sequence has located within the imperfect inverted repeat a G-N11-A instead of the predicted T. Mutagenesis of the G to a T resulted in an increase in both the binding of CatR to the catBC promoter and in the in vivo activation. Nucleotides within the -35 and -10 regions of the catBC promoter were found to be important for promoter activity but not for CatR binding.