Expression of the ada and alkA genes, both of which are involved in the adaptive response of Escherichia coli to alkylating agents, is positively controlled by Ada protein, the product of the ada gene. Large amounts of ada- and alkA-specific RNA were formed in cells treated with a methylating agent, whereas little such RNA was produced in untreated cells. The in vivo transcription-initiation sites for the two genes were determined by primer-extension cDNA synthesis. In an in vitro reconstituted system, both ada and alkA transcripts were formed in an Ada protein-dependent manner. However, responses of the two transcription reactions to methylating agents differed; ada transcription was stimulated by methylnitrosourea, while alkA transcription was suppressed. We prepared a methylated form of Ada protein by an in vitro reaction and compared the activity with that of the normal, unmethylated form. The methylated form was more effective in promoting ada transcription than was the unmethylated form, but the effects of both forms were much the same with regard to alkA transcription. Based on these findings, we propose a model for the molecular mechanism of adaptive response.