Many microorganisms exhibit an adaptive response to mutagenic alkylation damage. In Escherichia coli the response is regulated by the inducible Ada protein. A sensitive immunoassay employing two anti-Ada monoclonal antibodies has been developed here to monitor low levels of induction of the Ada protein. This protein was detected in non-induced E. coli which contained an average of two molecules of Ada per cell. The occurrence of the adaptive response in bacteria signals the existence of an ecological niche in which cells are exposed to direct-acting methylating compounds, but the structure and identity of these agents are unknown. Using the immunoassay to search for possible candidates, a number of methylating agents and precursors of such agents have been investigated. Carbamyl phosphate and methylamine yield N-methylurea, which reacts subsequently with nitrite to generate the strong inducer N-methyl-N-nitrosourea. The antibiotic streptozotocin also is a potent inducer of the adaptive response. Moreover, the abundant environmental mutagen methyl chloride acts as an inducer.