Prekallikrein was purified from human plasma with a final yield of 76% using as the principal step adsorption to immobilized chicken antikallikrein IgY. When purified prekallikrein (3.4 microM) was incubated in the presence of beta-Factor XIIa (0.068 microM) for 5 min at 37 degrees C and pH 7.5, alpha-kallikrein was obtained. Upon prolonged incubation (0.5-28 h), the Mr 52,000 heavy chain of alpha-kallikrein was progressively cleaved, resulting in the formation of beta-kallikrein. The formation of beta-kallikrein was characterized as an autolytic process because it was prevented by specific inhibitors of kallikrein, including aprotinin and antikallikrein antibody but not by corn trypsin inhibitor, an inhibitor specific for beta-Factor XIIa.