We have purified L-kynurenine 3-monooxygenase from pig liver mitochondria using a procedure involving seven steps composed of (1) preparation of mitochondrial outer membrane, (2) preparation of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) insoluble outer membrane material, (3) extraction of the enzyme with beta-octylglucoside, (4) ammonium sulfate fractionation, (5) DEAE-Sepharose CL-6B chromatography, (6) Matrex gel orange A affinity chromatography, and (7) high-performance liquid chromatography (HPLC) gel filtration. The final preparation had an about 160-fold purified enzyme activity with a yield of 0.8%. The apparent molecular mass of the aggregated form of the native enzyme was determined to be close to 300 kDa by HPLC gel filtration in the presence of 0.005% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a main protein band with an apparent molecular mass of about 49 kDa. The enzyme was found to be about 86% pure by the criterion of SDS-PAGE. The dissociated form of the enzyme contains 1 mol of non-covalently bound FAD/mol of protein monomer. The UV/visible spectrum had absorption peaks at 275, 384, and 450 nm, typical of a simple flavoprotein. Five inhibitory monoclonal antibodies against the enzyme were obtained. They could stain moderately a single protein band (49 kDa) in a Western blot.