We detected a prolyl endopeptidase (EC 3.4.21.26) in the cell-free extract of Pseudomonas sp. KU-22. The enzyme was purified approximately 2,000-fold by a series of column chromatographies. The molecular weight and isoelectric point of the purified enzyme were estimated to be 76,000 and pH 4.9, respectively. The enzyme was most active at 45 degrees and pH 8.0 with benzyloxycarbonyl-L-alanyl-L-alanyl-L-proline p-nitroanilide (Z-Ala-Ala-Pro-NphNO(2)) as a substrate. Significant inhibition of the enzyme by DFP and prolyl endopeptidase-specific inhibitors was observed. Some properties noted, particularly pH stability, amino acid composition, and pI of the enzyme, are clearly different from those of other bacterial prolyl endopeptidases reported to date, indicating that the enzyme is a member of the microbial prolyl endopeptidase family.