Lack of prolidase I (PD I) leads to prolidase deficiency, a disease characterized by intractable skin lesions, recurrent respiratory infections, and mental retardation. The present study was undertaken to characterize and determine the physiologic roles of different prolidase isoenzymes. Two isoforms of prolidase were isolated from rat kidney. PD I showed higher activity against seryl-proline and alanyl-proline, whereas PD II was active especially against methionyl-proline. PD I was highly concentrated in the small intestine and kidney, whereas PD II was shown not to vary in the organs examined. Expression of PD I and PD II in the small intestine were maximal within 1 wk of birth, and then rapidly declined. The changes of prolidase in the kidney and heart were found to differ slightly. N-benzyloxycarbonyl-l-proline and captopril inhibited PD I dose-dependently, but showed no inhibition of PD II at low concentrations. NiCl2 inhibited PD II much more effectively than PD I. Our findings suggest that PD I functions by way of an intestinal peptide carrier, which may also be regulated by the uptake of various iminodipeptides. Similarly, age-related alterations of prolidase isoenzymes suggest that intestinal PD II also participates in absorption of proline and other amino acids early in life.