Mucor rennin, a milk-clotting acid protease produced by a fungus Mucor pusillus, was inactivated by photo-oxidation mediated by methylene blue according to first order kinetics. The pH profile of the inactivation rate showed that a dissociating group with a pK value of 7.6 was involved in the inactivation. Addition of pepstatin A, an inhibitor specific for acid proteases, caused a marked alkaline shift of the pK value. One of two histidyl residues in the enzyme was destroyed by the photo-oxidation, with complete loss of the enzyme activity. Analysis of inhibitor binding activity and chemical modification with diazoacetyl-DL-norleucine suggested that the photo-oxidized enzyme still retained its original conformation. These results indicated that one histidyl residue in addition to the two essential carboxyl groups is involved in the catalytic function of Mucor rennin.