Prolidase I (EC 3.4.13.9) was purified to homogeneity from the erythrocytes of a normal human (control) and the patient's mother, and prolidase II from erythrocytes of a control and the patient's mother, and prolidase from the patient's erythrocytes was also highly purified. The various properties of the patient's prolidase were compared to those of prolidase from a control and the patient's mother. Prolidase I from a control and the patient's mother had a molecular weight of about 112,000, and was composed of two subunits with an identical molecular weight of 56,000. The Km values for Gly-Pro of the control's and the patient's mother's prolidase I were 2.90 +/- 0.22 and 2.88 +/- 0.27 mM, but the Vmax values for Gly-Pro of the mother's enzyme was reduced about 30% compared to that of control enzymes (mother: 6.02 units/mg protein, control: 22.21 units/mg protein). Isoionic points of these enzymes by chromatofocusing were pH 4.6 approximately 4.7. Prolidase II from the control and the patient's mother, and the patient's prolidase had a molecular weight of about 185,000, and was composed of two subunits with an identical molecular weight of 95,000. The Km and Vmax values for various substrates of prolidase II from a control and the patient's mother, and the patient's prolidase were almost the same.