Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline pK e (pK e1 and pK e2 ) values of about 4 and 10. In this study, we estimated the ionizable groups involved in its catalytic mechanism by thermodynamic analysis. pK a of side chains of L-Asp, L-Glu, L-His, L-Cys, L-Tyr, L-Lys, and L-Arg at 25–45°C were determined by the pH titration of amino-acid solutions, from which their enthalpy changes, ∆H°, of deprotonation were calculated. pK e1 and pK e2 of MMP-7 at 15–45°C were determined in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N 3 -(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH 2 , from which ∆H o for pK e1 and pK e2 was calculated. The ∆H o for pK e1 (−20.6±6.1kJmol −1 ) was similar to that for L-Glu (−23.6±5.8kJmol −1 ), and the ∆H o for pK e2 (89.9±4.0kJmol −1 ) was similar to those for L-Arg (87.6±5.5kJmol −1 ) and L-Lys (70.4±4.4kJmol −1 ). The mutation of the active-site residue Glu198 into Ala completely abolished the activity, suggesting that Glu198 is the ionizable group for pK e1 . On the other hand, no arginine or lysine residues are found in the active site of MMP-7. We proposed a possibility that a protein-bound water is the ionizable group for pK e2 .