Prolidase assay was reinvestigated by determining proline, using Chinard's method. Although several authors had previously tested this colorimetric reaction, accurate details regarding enzyme activity were not available. The need for greater sensitivity led to the introduction of several modifications: dialysis was eliminated and the substrate concentration and incubation time were changed. In addition, the reaction mixture was preincubated with Mn2+ for 24 h in order to triple prolidase activity. Color development followed at 90 degrees C, because of partial glycylproline hydrolysis at higher temperatures. The effect of several divalent cations on prolidase activity were tested with and without Mn2+. This modified assay was applied to erythrocytes, plasma and skin fibroblasts from a female patient with iminodipeptiduria.