Human plasma prekallikrein was purified from normal plasma. The purified prekallikrein appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave two protein bands with approximate Mr 85 000. Proteolytic activation of prekallikrein by purified human beta-factor XIIa (Mr 28 000 form) resulted in the formation of kallikrein. The apparent molecular weight of kallikrein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of mercaptoethanol was identical with that of prekallikrein; reduction of kallikrein yielded a heavy chain of Mr 52 000 and two light chains of Mr 42 000 and 37 000. The appearance of kallikrein activity was directly correlated with the limited proteolysis due to beta-factor XIIa. Kinetic and immunologic studies demonstrated that plasma prekallikrein is a factor XII dependent plasminogen proactivator. The rate constant for the inactivation of prekallikrein by diisopropyl phosphofluoridate was similar to that previously reported for trypsinogen. This observation raises the possibility that low intrinsinc catalytic activity of prekallikrein may play a role in the initiation of the intrinsic blood coagulation pathway.