The adaptive response of Escherichia coli protects the cells against the toxic and mutagenic effects of certain alkylating agents. The major effector molecule regulating this response is the 39-kDa Ada protein, which functions as both a DNA repair protein and a transcriptional activator. Ada removes methyl groups from phosphotriester and O6-methylguanine lesions in DNA, irreversibly transferring them to cysteine residues at positions 69 and 321, respectively. When methylated at Cys-69, Ada is converted into a potent activator of ada and alkA transcription and binds to a sequence (Ada box) present in both promoters. We have found that physiologically relevant higher concentrations of unmethylated Ada are able to inhibit the activation of ada transcription by methylated Ada, both in vitro and in vivo. In contrast, the same concentrations of unmethylated Ada do not inhibit the activation of alkA transcription by methylated Ada, either in vitro or in vivo. Deletion of the carboxyl-terminal 67 amino acids of Ada abolished the ability of the unmethylated form of the protein to inhibit activation of ada transcription but not the ability of the methylated form to activate ada or alkA transcription. Our results suggest that the Ada protein plays a pivotal role in the negative modulation of its own synthesis and therefore in the down-regulation of the adaptive response. Elements present in the carboxyl terminus of Ada appear to be necessary for this negative regulatory function.