The biological effects of alkylating agents in both pro- and eukaryotes are thought to be mediated via alkylation at the O ⁶ -position of guanine in DNA (1–4). Repair of such adducts can be mediated by O ⁶ -alkylguanine-DNA alkyltransferase (ATase; 3,4). Both pro- and eukaryote ATases transfer alkyl groups from the O ⁶ -position of guanine in alkylated DNA (or from other low-molecular-weight substrates) (5) to a cysteine residue located at the active site of the protein: the reaction is stoichiometric and the protein is autoinactivated (6). This mechanism has been exploited in the design of several different radioactivity-based assays for the enzyme. These involve either measurement of methyl group transfer to protein or the analysis [e.g. by high-performance liquid chromatography (HPLC)] of methylated substrate DNA before and after exposure to cell or tissue extracts or restriction endonuclease (RE) site deprotection of synthetic oligonucleotide substrates containing O ⁶ -methylguanine.