The proteolytic regulation of peptides involved in feeding behavior is poorly understood.Prolyl carboxypeptidase (PRCP) is particularly known for its role in body weight control by converting the anorexigenic peptide, α-MSH 1-13, into the inactive 1-12 form.The purpose of this study was to characterize purified human PRCP, to investigate its substrate specificity and to discover novel substrates linked to obesity.Pyroglutamated apelin-13, ghrelin, enterostatin and obestatin were investigated since these are feeding-regulating peptides with potential cleaving sites for PRCP.PRCP was purified 575-fold from human placenta and successfully identified as human lysosomal Pro-X carboxypeptidase.The kinetic parameters of purified and com. available PRCP for known and potential peptide substrates were determined and compared using a RP-HPLC activity assay, isothermal titration calorimetry, and mass spectrometry.Purified and recombinant PRCP had similar substrate specificities with angiotensin III as the substrate of preference.Pyroglutamated apelin-13 was observed to be a novel substrate for human PRCP in vitro and PRCP-dependent cleavage was shown in a human umbilical vein endothelial cell culture experimentOther potential substrates, e.g., obestatin, ghrelin, and enterostatin, were not hydrolyzed by PRCP.These results showed that the placenta is a good source of human PRCP and that PRCP removes the C-terminal phenylalanine from pyroglutamated apelin-13.Thus, for the 1st time, PRCP is identified as an apelin-cleaving enzyme.This finding adds evidence to the hypothesis that PRCP plays a role in energy homeostasis.