Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretically to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yield of 9 % and a specific activity of 187.4 U/mg protein. The native enzyme (molecular weight, 280,000) was composed of eight identical subunits with apparent molecular weights of 35,000. The cephalosporin-C deacetylase was stable up to 60-degrees-C for 30 min at pH 7.0. The enzyme exhibited Michaelis-Menten kinetics with the substrates cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and p-nitrophenyl acetate; the K(m) values were 24.0, 7.9 and 1.0 mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak noncompetitive inhibitor and other product, acetate, was a weak competitive inhibitor; the K(i) values were 171 and 290 mM, respectively. However, these weak product inhibitors did not prevent the completion of the deacetylation of 7-ACA. The pI value of the enzyme was determined to be 5.3 using isoelectric focusing. The observed data indicate that the enzyme is different from known cephalosporin-C deacetylases. In addition, amino acid sequencing of the N-terminus and Achromobacter proteinase 1-digested peptides yielded no sequences with similarities to other known proteins by a computer search.