A protease in the venom of Ophiophagus hannah (king cobra) has been purified to a homogeneous state by successive chromatographies on Sephadex G-100 superfine, DEAE-cellulose, hydroxyapatite and CM-polyvinylalcohol copolymer columns. The mol.wt as determined by SDS-PAGE and gel filtration was approximately 70,000. The purified enzyme possessed a specific activity approximately 1/25 that of crystalline trypsin, whereas it had no hemorrhagic activity. The substrate specificity was determined using oxidized insulin B-chain as a substrate; the enzyme cleaved the Asn3-Gln4, Gln4-His5, His10-Leu11, Ala14-Leu15 and Tyr16-Leu17 positions. The sites cleaved by the protease were compared to proteases from other snake venoms.