Human paracaspase has been predicted to be a member of the protein structural fold that encompasses protease clan CD. To determine whether paracaspase has catalytic activity we have expressed the region corresponding to the catalytic domain and used protease activity-based chemical probes to profile the putative active site. A leucine-based acyloxymethyl ketone probe that covalently labels cysteine proteases discloses a hydrophobic P 1 preference in the putative active site. The probe covalently labels Cys539, which is not the predicted catalytic site based on structural and sequence comparisons with other clan CD proteases. Using a combinatorial peptide substrate library approach we have been unable to detect amidolytic activity of paracaspase, implying that if it is a protease it must be very specific. We suggest a switch in the use of catalytic residues to generate an enzyme overlapping the canonical clan CD protease active site.