Kynureninase [L-kynurenine hydrolase EC 126.96.36.199] has been purified 614-fold from rat liver cytosol. The purification procedure involved pH treatment, ammonium sulfate fractionation, Sephadex G-100 gel filtration, DEAE-Sepharose CL-6B chromatography, extraction with 40% saturated solution of ammonium sulfate and hydroxyapatite chromatography. The enzyme was found to be homogeneous by the criteria of disc gel electrophoresis. SDS-gel electrophoresis and sucrose gradient centrifugation. The enzyme was obtained as a holo-enzyme which showed an absorption maximum at 420 nm. In the absence of pyridoxal 5-phosphate (PLP) the enzyme was dissociated into an apoenzyme. The isoelectric points of holoenzyme and apoenzyme are 5.7 and 6.1. Sucrose gradient centrifugation and SDS-gel electrophoresis gave molecular weight estimates of 95,000 and 55,000, respectively. The optimum pH shifted to higher pH with increase in the concentration of PLP. The Michaelis constants were determined as follows: kynurenine, 240 microM; 3-hydroxykynurenine, 13 microM; PLP, 0.1-1.7 microM. The maximum velocity for 3-hydroxykynurenine was 11 times higher than that for kynurenine at pH 7.7. This enzyme can be regarded as a 3-hydroxykynureninase type enzyme.