The study of a novel prolme-specific peptidase from bovine serum is presented. The enzyme readily cleaves Z-Gly-Pro-MCA liberating the fluorophore MCA thus allowing quantification of enzyme activity Unlike prolyl ohgopeptidase (PO) which also hydrolyses this fluorogemc substrate, this peptidase is completely insensitive to Z-Pro- Prolmal and has been designated Z-Pro-Prolmal Insensitive Z-Gly-Pro-MCA degrading Peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl sepharose, calcium phosphate cellulose and DEAE anion exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023 SDS PAGE and size-exclusion chromatography indicated a heterodimeric structure with a relative molecular mass of 180kDa. The enzyme remained stable at temperatures less than 50X1 for up to an hour, while optimal activity was observed at 37°C ZIP was surprisingly stable over the pH range 2 5-JO 0 Optimal activity was detected in the range pH 7 4-8 0 Isoelectric focusing revealed a pi value of5 68 and the enzyme appears to be 33% glycosylated. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site a2Mfailed to inhibit activity suggesting an ohgopeptidase specificity HPLC analysis revealed a broad substrate specificity for proline-containmg peptides Kinetic analysis indicated that ZIP had a greater affinity for Z-Gly-Pro-MCA than PO with a Km of 54pM deduced. The ohgopeptidase showed complete insensitivity to a number of PO-specific inhibitors, namely JTP-4819 and S- 17092-1. ZIP exhibits similar biophysical characteristics to PO isolated from a number of sources. However the peptidases do appear to be distinct and ZIP may represent a novel prohnespecific serum peptidase, which may play a role in the degradation of oligopeptides.