Activation of prorenin in the neutral phase after pH 3.3 dialysis of human plasma depends on clotting factor XII-initiated prekallikrein to kallikrein conversion. Acid dialysis may be necessary for destroying kallikrein inhibitors or rendering prorenin susceptible to attack by kallikrein. If the latter possibility proves true, it is difficult to see how the factor XII-kallikrein pathway could activate prorenin in vivo. Plasma prorenin was therefore separated from active renin and from the protease inhibitors alpha 2-macroglobulin, C1-inactivator, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, and antithrombin III by gel filtration on Sephadex G-100 and affinity chromatography on Blue Sepharose CL-6B at neutral pH. The resulting prorenin preparation could be activated at pH 7.5 by highly purified human plasma kallikrein, which was prepared from prekallikrein by activation with active factor XII fragment beta-factor XII a. Activation proceeded at 4 and 37 C at a kallikrein concentration of 2 micrograms/ml, which is approximately 5% of the prekallikrein concentration in normal plasma. It appears that an acid-induced conformational change of the prorenin molecule is not required for its activation by plasma kallikrein.