A chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P-1 Leu, Met, Phe, Trp, and Tyr appearing in other chymotrypsin inhibitors, a P-1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 muM) with chymotrypsin. Phylogenetic; analysis suggests that the functional variations of the chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.