Abstract:A cysteine endopeptidase (EC 3.4.22) was purified from cotyledons of germinated peanut seeds by buffer extraCtion, ammonium sulfate precipitation and succesive chromatagrafies on Sephadex G-100, DEAE-cellulose and DEAE-Sephedax A-50 columes. The overall purification and final recovery were 28.1 fold and 15.9%. This endopeptidase exists in two isoenzymic forms and SDS-PAGE revealed two polypeptides of 58 and 55KD. The optimumtemperature was 50℃ at 8.1 pH for degrating BAPNA (Benzoylarginine-p-nitroanilide) in Tris-HCl buffer. Inhibitor studies demonstrated that the endopeptidase belongs to the cysteine class of endopeptidase. This enzyme hydrolysed only 5-10% of arachin or coarachin Ⅰor 2s protein of ungerminated peanut seeds in vitro. The characterization of the endopeptidase was almost consistent with the peptide hydrolase in the study of Cameron and Mazelis who purificated from cotyledons of ungeednated peanut seeds.The results of present paper and our other studies suggested that the cysteine endopeptidase of peanut cotyledons which plays an important role during seed germination was synthesized during seed development and can only degrade the modified storge proteins.