Prolyl oligopeptidase is a serine peptidase characterised by oligoendopeptidase activity. Definitive evidence for the discrete biological role of prolyl oligopeptidase remains unknown, though its role in the maturation and degradation of peptide hormones and neuropeptides has been implicated. A second activity that readily cleaves the specific prolyl oligopeptidase substrate Z-Gly-Pro-AMC was previously discovered in bovine serum. Due to its complete insensitivity towards the classic prolyl oligopeptidase inhibitor Z-Pro-prolinal, this peptidase was designated Z-Proprolinal Insensitive Peptidase (ZIP). The study o f this new and specific proline cleaving endopeptidase from bovine serum is presented. ZIP was separated from prolyl oligopeptidase by phenyl sepharose hydrophobic interaction chromatography. The enzyme was further purified 30,197-fold, to homogeneity, using calcium phosphate cellulose, cibacron blue 3GA and gel filtration chromatography in an overall recovery o f 12% from bovine serum. Substrate specificity studies using kinetic, HPLC and LC-MS analysis of prolinecontaining peptides suggest that ZIP has an extended substrate-binding region in addition to the primary specificity site Sj. This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position Pi was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end o f the scissile bond (Pi) was evident. An affinity constant (Kn) o f270fM using Z-Gly-Pro-AMC was determined. Diisopropylfluorophosphate inactivated ZIP resulting in an IC$o value o f lOOnM suggesting catalytic classification as a serine peptidase. ZIP showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, Fmoc-Ala-pyrrCN and ZPhe-Pro-BT and showed no immunological cross-reactivity with an anti-human prolyl oligopeptidase antibody. Internal peptide sequence analysis o f ZIP identified it as seprase, a member o f the serine integral membrane peptidases. This protein is overexpressed by tumour cells and may possibly play a significant role in their invasion.