Selenosubtilisin, a semisynthetic enzyme produced by chemical modification of subtilisin's catalytic serine, mimics the antioxidant enzyme glutathione peroxidase, catalyzing the reduction of hydroperoxides by 3-carboxy-4-nitrobenzenethiol. In analogy with the natural peroxidase, a variety of hydroperoxides are accepted as substrates for the semisynthetic enzyme, whereas the dialkyl compound tert-butyl peroxide is not. Kinetic investigations reveal that kmax is dependent upon the nature of the hydroperoxide, indicating that peroxide-mediated oxidation of the enzymic selenolate is at least partially rate-limiting. Experiments with the radical trap 2,6-di-tert-butyl-4-methylphenol suggest that, while the nonenzymic reaction between tert-butyl hydroperoxide and thiol involves free radicals, the same reaction catalyzed by selenosubtilisin does not. The studies described here support the enzyme's proposed ping-pong mechanism and are consistent with previous mechanistic observations.