Cbz-Phe-epoxide was designed as a selective inactivator of cysteine proteases. It exhibits a time- and concentration-dependent inactivation of cysteine proteases, while showing no activity towards serine proteases. The inhibition is irreversible, correlated with loss of the free active-site thiol, and its rate is at least 10(4) faster than the rate of a model reaction in solution. These results support the proposed active-site directed, protonation-dependent, mechanism-based mode of inactivation of cysteine proteases by the new inhibitor.