The effect of various amino acids and MnCl 2 on prolinase activity in erythrocyte lysates from a healthy individual and a patient with prolidase deficiency was investigated. A concentration of 0.1 mM MnCl 2 increased prolinase activity in normal erythrocytes against pro-gly, pro-glu, pro-leu, pro-ser and pro-phe, but inhibited that against pro-ala, pro-val, pro-met and pro-asp. However, prolinase activity against these iminodipeptides was enhanced by pre-incubation with glycine, independent of MnCl 2 . The same studies on erythrocytes from a prolidase-deficient patient showed almost the same results as the normal control, except that prolinase activity against pro-gly and pro-ser was slightly inhibited by adding 0.1 mM MnCl 2 . Some amino acids, glutamic acid and glutamine, slightly enhanced prolinase activity against progly in erythrocytes from both the normal control and the prolidase-deficient patient, but N-acetyl-L-glutamic acid, γ-aminobutyric acid (GABA) and β-alanine showed no effect. Branched amino acids, L-valine, L-leucine and L-isoleucine strongly inhibited the prolinase activity against pro-gly. However, conversely, their isomers, D-valine, D-leucine and D-isoleucine, enhanced it. The kinetics of prolinase activity in the erythrocytes from both the normal individual and the prolidase-deficient patient were also studied. Their K m values were changed by adding glycine or 0.1 mM MnCl 2 , but V max values were almost the same.