The inactivation of highly purified papain (2 × 10−5 M in distilled water) by hydrogen peroxide, generated by the γ-irradiation of water, was examined. The kinetics of activity loss at 23 °C was second order (k = 3.7 × 103 M−1 min−1) for papain:peroxide molar ratios of 1:1 or 2:1. Loss of activity is accompanied by a parallel loss of sulfhydryl; however, the sulfhydryl losses, as determined with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) or p-hydroxymercuribenzoate (pHMB), are anomalously either too large or too small, respectively. These discrepancies resulted from the reaction of inactive papain with either the thiol anion product of the DTNB reaction, or with the pHMB reagent itself. The addition of 1.2 M urea to the DTNB reaction mixture significantly decreased this error. Inactive papain reacted with high concentrations of cysteine or cyanide to yield completely repaired active papain, and with benzylamine to yield non-repairable, inactive papain. Sodium arsenite, which is capable of reducing sulfenic acids but not disulfide bonds, readily repaired peroxide-inactivated papain. A completely inactive but repairable papain fraction was isolated by virtue of its lessened ability to bind to a tetrapeptide inhibitor immobilized on Sepharose. The cumulative results indicate that the peroxide inactivation of papain is due almost exclusively to the formation of papain sulfenic acid (Cys25—SOH).