1. Prolidase I (EC 3.4.13.9) was purified from human kidney to SDS-PAGE homogeneity. The molecular weights of native and denatured purified enzyme were estimated to be 115,000 and 55,000, respectively. 2. Agarose electrophoresis revealed migration in the alpha 1 globulin region, and an isoelectric point (pl) of 4.65 was estimated by both isoelectric focusing (IEF) and the titration curve method. 3. Activation by preincubation for 24 hr at 37 degrees C with 1 mM MnCl2 was maintained throughout the purification steps, using gly-pro and phe-pro dipeptides as substrates. 4. Activation in the presence of gly-pro was higher (4.5- to 11-fold) than in the presence of phe-pro (1.3- to 2.3-fold). 5. Lineweaver-Burk plot consisted of one and two lines with gly-pro and phe-pro, respectively. Km, Vmax and the Vmax/Km ratio were increased and the two lines with phe-pro were conserved after prolonged preincubation. 6. A specific polyclonal antibody was raised in rabbits against the purified enzyme and immunoreactivity was investigated between rabbit antiserum and both prolidase I from various tissues and human kidney prolidase II. 7. Prolidase I from liver, erythrocytes and plasma was immunochemically identical to renal prolidase I. The polyclonal antibody did not react with prolidase II. 8. These results indicate that a specific immunoassay might be developed to investigate prolidase I protein in plasma and tissues from patients with prolidase deficiency and hepatic fibrosis.