DNA fragments of Bacillus subtilis were inserted into a plasmid vector that can multiply in Escherichia coli cells, and foreign genes were expressed under the control of the lac promoter. By selecting hybrid plasmids that confer an increased resistance to alkylating agents on E. coli ada- mutant cells, the B. subtilis gene dat, which encodes O6-methylguanine-DNA methyltransferase, was cloned. The Dat protein, with a molecular weight of about 20,000, could transfer the methyl group from methylated DNA to its own protein molecule. Based on the nucleotide sequence of the gene, it was deduced that the protein comprises 165 amino acids and that the molecular weight is 18,779. The presumptive amino acid sequence of Dat protein is homologous to the sequences of the E. coli Ogt protein and the C-terminal half of the Ada protein, both of which carry O6-methylguanine-DNA methyltransferase activity. The pentaamino acid sequence Pro-Cys-His-Arg-Val, the cysteine residue of which is the methyl acceptor site in Ada protein, was conserved in the 3 methyltransferase proteins. The structural similarity of these methyltransferases suggests possible evolution from a single ancestral gene.