The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-- > A:T as well as A:T-- > G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.