A /Mactamasewas purified from Klebsiella oxytoca strain GN10650. The enzymewas chromosomally-mediated and gave a single protein band on polyacrylamide gel electrophoresis. Its pi was 5.34 and its MWwas approximately 27,000. The optimal pH and temperature were about 7.0 and 50°C, respectively. The specific activity of the enzyme was 1,207 units per mgof protein for hydrolysis of penicillins and cephalosporins, including cefuroxime, cefotaxime, and aztreonam. The enzyme activity was inhibited by /?-chloromercuribenzoate, iodine, ferrous ion, and by clavulanic acid. Rabbit antibodies raised against the purified K. oxytoca enzymeshowedno cross-reactivity in neutralization tests with /Mactamases produced by other species of Gram-negative bacteria.