The cytotoxic alkylation lesions 1-methyladenine (1-alkyladenine) and 3-methylcytosine are removed efficiently from DNA by direct damage reversal, catalyzed by the Escherichia coli AlkB protein and its human homologs ABH2 and ABH3. The enzymes act by oxidative demethylation, employing Fe(II) and alpha-ketoglutarate as cofactors, and release the methyl moiety as formaldehyde. The isolation of these enzymes from overproducing cells is described, as well as the preparation of radioactively labeled substrates and procedures for enzyme assays. Functionality in vivo is examined by complementation of the low survival of alkylated single-stranded DNA bacteriophage in an E. coli alkB mutant.