The location and state of an essential histidyl residue in a milk-clotting acid proteases, Mucor rennin, were investigated by NMR spectroscopy. Assignment of the C 2 H resonance peak of the essential histidyl residue was possible by comparison of the NMR spectrum of the native enzyme with that of the photo-oxidized enzyme. The pH titration curve for the chemical shift of the C 2 H proton showed two inflections, a major one with pK_a=7.4 and a minor one with pK_a =3.5 at 30°C. The major inflection, corresponding to an intrinsic protonation of the imidazole ring, shifted toward the alkaline side upon addition of acetyl pepstatin, an inhibitor specific for the acid protease. Modification of an essential carboxyl group in the enzyme with diazoacetyl-DL-norleucine caused disappearance of the minor inflection as well as an acidic shift of the major pK_a value. Perturbation effects on the C 2 H resonance of the lanthanide metals, Pr³⁺, Eu³⁺, and Gd³⁺, suggested their selective binding to a carboxyl group and location of the bound metal atom close to the essential histidyl residue. All data suggested that the essential histidyl residue of Mucor rennin is located close to one of the two essential carboxyl groups in the catalytic site of the enzyme.