3-hydroxykynureninase in human liver was present in cytosol and mitochondria. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent Km values of the enzyme were 7.7 X 10(-5) M for 3-hydroxykynurenine, 1.0 X 10(-3) M for kynurenine and 2.5 X 10(-6) M for pyridoxal 5'-phosphate with 3-hydroxykynurenine. Some other properties of purified enzymes are described.