Papain (E.C. 3.4.22.2), a cysteine protease in papaya, was activated with reducing agents and treated with pulsed electric fields (PEF) in a continuous system at 10°C. Irreversible reduction of activity was observed in PEF-treated papain after 24 h storage at 4°C. Oxidation of papain active site, a cyteine residue, was not the major cause of papain inactivation by PEF. Temperature did not increase over 35°C during PEF treatment. The pH of papain solution was not changed after PEF treatment. Structural change was observed in PEF-treated papain by CD analysis. Inactivation of PEF-treated papain was related to the loss of α-helix structure. Heating at 60-80°C for 2 min did not significantly reduce the activity of papain.