Based on the liberation of proline from ProLeuGlyNH2 (MIF-1, melanostatin) manganese-activated prolyl aminopeptidase activities were purified from rat brain and kidney cytosolic fractions. They were distinguished from other di- and tripeptidases and an arylamidase liberating N-terminal proline. Purified prolyl aminopeptidase from both sources had identical molecular properties (native Mr 300,000, subunit Mr 54,000) and very similar catalytic properties. The action of the purified enzymes was not restricted to proline. Other, in particular lipophilic, amino acids were cleaved from di-, tri- and oligopeptides with even higher velocities. Peptides with N-terminal penultimate proline residues were not degraded. From a comparison of molecular data, action on peptides, influence of pH values, inhibitors and activators, it is concluded that the activity is identical with leucyl aminopeptidase (EC 3.4.11.1) and that a separate prolyl aminopeptidase (EC 3.4.11.5) does not exist in rats.