Low concentrations of KCNO (approx o.1. mM) were found to react rapidly with activated papain (EC 188.8.131.52), leading to inactivation of the enzyme. The reaction proved to be reversible, the activity slowly returning again on sufficient dilution of the enzyme-KCNO mixture. The reaction occurred with the essential thiol group of papain, shown by an emperometric method. The rate of inactivation could be conveniently determined from the shape of the progress curves of ester hydrolysis (5–120 mM benzoylarginine ethyl ester), traced on the recorder of a pH stat. The rate constant k was found to vary with substrate concentration. It decreased with increasing sustrate concentration. A plot was made of k versus [E]/([E] + [ES]) as calculated from the known value of Km. A linear relationship was found, which gave extrapolated values for k = 9400 M-1 · min-1, at zero substrate concentration and of k = 1500 M-1 · min-1 at infinite substrate concentration. The latter value shows that protection of the enzyme in the ES complex is not complete. The rate constant of the reaction of the thiol group of free cysteine with KCNO under the same conditions (pH 6.0; 25°) was only 3.4 M-1 · min-1. Hence the reactivity of cyanate towards the thiol group of papain, as determined by extrapolation to zero substrate concentration, is 3000 times greater than the reactivity of the thiol group of free cysteine.