The adaptive response is a cellular process to induce DNA repair enzymes in response to a challenge of alkylating agents. In this process Ada protein, the product of the ada gene, plays a major role; it accepts the methyl groups of the methylated DNA at the cysteine residues of its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, thereby triggering induction of the whole process. In addition to this DNA-mediated activation of Ada protein, we have proposed an alternative mechanism which involves direct methylation of Ada protein by methylating agents (Takahashi, K., and Kawazoe, Y. (1987) Biochem. Biophys. Res. Commun. 144, 447-453). Here we present evidence that Ada protein is activated as a transcriptional regulator through a direct methylation by certain methylating agents. A purified preparation of Ada protein was treated with various methylating agents, and the activity to promote transcription of the ada gene was determined using an in vitro reconstituted system. The ada-specific RNA was produced when the Ada protein treated with methyl methanesulfonate or with methyl iodide was present in the reaction mixture. The Ada protein treated with N-methyl-N-nitrosourea did not show such an activity. It is, therefore, suggested that the adaptive response induced by chemoselective methylating agents such as methyl iodide might be due, at least in part, to the direct methylation of the constitutive Ada protein present in the cell.