The ada+ gene of E. coli is a regulatory gene of the adaptive response to simple alkylating agents. ada mutants are sensitive to both the mutagenicity and toxicity of alkylating agents, and are unable to induce O6-methylguanine DNA methyltransferase and 3-methyladenine DNA glycosylase II. The ada+ gene was cloned from wild type E. coli B by ligating bacterial DNA partially digested with Sau3A into the cosmid vector pJB8. The hybrid cosmid, pCS33, conveyed N-methyl-N'-nitro-N-nitrosoguanidine resistance to ada mutants of E. coli B and E. coli K12, and resulted in the constitutive synthesis of the two DNA repair enzymes at high levels. An alk mutation, which results in a deficiency of only the DNA glycosylase, was not complemented by this cosmid. It was concluded that the product of the ada+ gene is a positive regulator of the adaptive response. The cosmid insert DNA was subcloned into the plasmid vector pAT153, and the ada+ plasmids pCS42 and pCS58 selected. The ada+ gene was located in pCS58 by transposon mutagenesis and subcloning. Two polypeptides of Mr 37,000 and 27,000, were identified in 'maxi-cells' as products of the ada+ gene(s). It is as yet unclear whether they represent different forms of the same gene product, or are encoded by separate ada+ genes within the same operon.