The rate of activation of papain (EC 220.127.116.11) by cysteine was studied in the presence of various concentrations of a substrate, benzoylarginine ethyl ester. The activation rate was found to decrease with increasing substrate concentration. This was interpreted as due to binding of the substrate to the non-activated enzyme. From the data obtained, the dissociation constant Ks of the complex of substrate and non-activated papain could be calculated. Ks was found to be virtually equal to Km, the Michaelis-Menten constant of the active enzyme, both at pH 6 and pH 4. One of the conclusions of this investigation is that the thiol group of active papain is not essential for substrate binding.