Abstract The effect of prolonged preincubation for 24 h at 37° C in the presence of 1 mmol/l manganese at pH 7.8 was investigated on the two forms of human erythrocyte prolidase after their separation by DEAE-Sephadex chromatography. Prolidase I activity, which was eluted in Chromatographic fractions of low ionic strength of about 160 mmol/l NaCl, increased after preincubation and this activation was maintained throughout the subsequent steps of chromatofocusing, chromatography on Sephacryl S-200 in tandem with Blue-Sepharose, and Phenyl-Sepharose chromatography. It disappeared after the last step consisting of a second Blue-Sepharose chromatography. When the protein environment was restored, isolated prolidase I was reactivated by preincubation, and as a result, gly-pro procollagen dipeptide became the best substrate. Gly-pro-hyp procollagen tripeptide was also observed to have a strong activator effect. The activity of prolidase II, which was eluted in high ionic strength fractions of about 260 mmol/l NaCl during DEAE-Sephadex chromatography, diminished markedly after preincubation and was very low against the gly-pro substrate. These results seem to indicate that prolidase I is much more active than prolidase II in the intracellular degradation of procollagen.