We describe prolinase (EC 3.4.13.8) activity in human plasma for the first time. Optimum activity was obtained with prolylvaline as substrate and 0.02 mmol/L manganese concentration at pH 9.0. Moreover, preincubation with manganese was not required, contrary to prolidase (EC 3.4.13.9) activity. The mean value observed in 106 subjects without liver and renal disorders was 16 U/L +/- 14 (2 SD). We determined this plasma enzyme activity in patients with acute hepatitis and chronic liver disease. Plasma prolinase activity was strongly dependent upon cytolysis because of the high activity in liver and the low activity in plasma. Of 24 patients with chronic liver disease (4 chronic hepatitis and 20 cirrhosis) and without cytolysis, prolinase activity was slightly increased in only three patients, whereas prolidase activity was increased in 13. This could be due to a difference in the activation of these two enzymes in liver during the fibrotic process.