Through the screening of yeasts and bacteria, some strains of Agrobacterium radiobacter have been found to efficiently concert 7-aminocephalosporanic acid (7-ACA) to deacetyl 7-ACA. The enzyme responsible for the conversion was constitutively formed by the bacterium. The 7-ACA deacetylating enzyme was purified from a cell-free extract of A. radiobacter IFO 12607 to homogeneity. The purified enzyme was a tetramer, composed of identical subunits with a molecular mass of 26 kDa, and showed activity toward some derivatives of 7-ACA, aryl acetates, monoacetin and triacetin, but was not active towards alkyl acetates and cephalosporin C. The K-m and k(cat) values of the enzyme for 7-ACA were 8.24 mM and 81 s(-1), respectively. Serine inhibitors and sulfhydryl reagents were both found to inhibit the activity. An amino acid sequence, GDSLT, which is the active center motif of an arylesterase, was identified in the amino terminal region. These results indicate that the enzyme belongs to the family of arylesterase (EC 3.1.1.2), and is a new 7-ACA-deacetylating enzyme.