cDNA encoding rabbit O6-methylguanine-DNA methyltransferase that repairs DNA damaged by alkylating agents was isolated, using as a probe a fragment of mouse cDNA coding for a region containing the active site of the enzyme. The nucleotide sequence of the cDNA revealed that the rabbit methyltransferase is a 181-amino acid polypeptide with a mol. wt of 19,385. Expression of the cDNA in a methyltransferase-deficient Escherichia coli mutant resulted in appearance of a 23 kDa polypeptide with methyltransferase activity, and this rendered the E.coli cells resistant to N-methyl-N'-nitro-N-nitrosoguanidine, in terms of both cell killing and induction of mutation. The rabbit methyltransferase is highly homologous to this enzyme in human, mouse, rat and hamster, but is 26-30 amino acid residues shorter as compared with methyltransferases in other mammalian species. Based on a comparison of the nucleotide sequences for the C-terminal regions of these proteins, we propose that a single base substitution, which would generate a TGA termination codon, was introduced into the sequence for the rabbit enzyme during the process of evolution. Existence of the naturally occurring truncated form of methyltransferase suggests that the longer C-terminal tails of other mammalian methyltransferases may have no significant role in exerting functions of the enzyme in vivo.