1. When Bacillus cereus 569/H was grown in a casamino acid (casein-hydrolysate) medium containing zinc sulphate rapid production of extracellular β-lactamase II preceded that of β-lactamase I. 2. β-Lactamase I was separated from β-lactamase II by fractional precipitation with ammonium sulphate. 3. β-Lactamase I was purified by a process involving chromatography on Celite and DEAE-cellulose and β-lactamase II by chromatography on DEAE-cellulose after denaturation of β-lactamase I by heat. Both enzymes were obtained in crystalline form. 4. β-Lactamase II prepared in this way appeared to have a higher molecular weight than β-lactamase I and required Zn2+ as a cofactor for both cephalosporinase and penicillinase activities.
