The biochemical mechanism and developmental expression for the repair of alkylated DNA has been characterized from Drosophila. As in other organisms, the correction of O6-methylguanine in Drosophila was found to involve the transfer of a methyl group from DNA to a protein cysteine residue. Two methylated proteins with subunit molecular weights of 30 kDa and 19 kDa were identified following incubation with [3H]-methylated substrate DNA and denaturing polyacrylamide gel electrophoresis. Identical molecular weights were found for the unmethylated forms of protein through their reaction to an antibody prepared against the 19 kDa Escherichia coli methyltransferase. Both Drosophila proteins are serologically reactive in adult males and females and most of the other developmental stages tested, with embryos representing the possible exception. The Drosophila proteins do not appear to be induced by sublethal exposures to alkylating agent.