A novel arylesterase from Agrobacterium radiobacter IFO 12607 catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA) to form deacetyl 7-ACA, but is inactive with cephalosporin C. A DNA fragment carrying the gene encoding the 7-ACA-deacetylating enzyme was cloned from the chromosomal DNA of this bacterium. The open reading frame encoding the enzyme was 642 bp long, corresponding to a protein of 214 amino acid residues (molecular mass=23,085). The deduced amino acid sequence did not contain the sequence GXSXG, typical of the many serine esterases including Bacillus cephalosporin C deacetylase, but has the pentapeptide motif sequence GDSLT (amino acid position 9-13) which is also a consensus sequence of some serine esterases. The newly cloned gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product purified from E. coli exhibited the same catalytic properties as the enzyme purified from A. radiobacter. Site-directed mutagenesis of S11A or S11C within the pentapeptide motif sequence led to complete loss of the enzyme activity. Thus, the Ser-ll residue within the GDSLT motif sequence was determined to construct the catalytic center. These results together with those of our previous studies indicated that the 7-ACA-deacetylating enzyme from A. radiobacter IFO 12607 is a new member of the family of lipolytic serine esterases containing the GDSLT sequence as their catalytic center.