The plasmid pO61 that was isolated from an E. coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E. coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e. similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein. A different set of hybridisation signals was produced when E. coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E. coli. The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.