Peptides containing aziridine-2,3-dicarboxylate (Azi) as electrophilic building block are evaluated as inhibitors of the cysteine proteases papain, cathepsin B, cathepsin L and clostripain. The influence of a free carboxylic acid as functional group at different positions of the inhibitor molecule on inhibition is analyzed. Structure-activity relationships and binding mode hypotheses are discussed. In contrast to the bacterial enzyme clostripain, the papain like mammalian proteases (cathepsins) are irreversibly inactivated by aziridinyl peptides. N-Unsubstituted aziridines are much more potent inhibitors of papain and cathepsins if they contain the free carboxylic acid attached to the aziridine ring (HOAzi-Leu-ProOBzl). Two free carboxylic acid functions at the aziridine ring are necessary for good inhibition of these enzymes by N-acylated aziridinyl peptides (BOC-Phe-Azi(OH)2). Chimeric bispeptidyl derivatives are selective CB inhibitors if the free acid is located at the C-terminus of the peptide (BOC-Phe-(EtO)Azi-Leu-ProOH). Clostripain is only inhibited by aziridinyl peptide esters.