A new kallikrein inhibitor in human skin extract was further purified by successive column chromatography on DEAE-cellulose, hydroxylapatite-cellulose, and p-cellulose. By these procedures, 0.7 mg of purified preparation was obtained from 10 g of original skin. The purified material was homogeneous, as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugation. It had an S20, w value of 4.2 and an apparent molecular weight of 57,000 as measured by gel filtration on Sephadex G-200, and was heat unstable. It possessed an inhibitory activity towards not only human plasma kallikrein, but also human urinary and pancreas kallikrein. It also exhibits antiplasmin and antithrombin activity. This kallikrein inhibitor was found to be immunologically distinct from α2-macroglobulin, α1-antitrypsin, or Cl-inhibitor.