The maximal activity of the dihydrothymine dehydrogenase of fractions of human blood was found in leukocytes, especially in the supernatant after centrifugation of homogenates at 100,000 × g in the course of isolation of the cell components. The dihydrothymine dehydrogenase from human and pig leukocytes was purified tenfold with a yield of activity of about 60%. Gel filtration with Sephadex G-200 showed several peaks of enzymatic activity. A quantitative method for the estimation of thymine and dihydrothymine by means of thin layer chromatography and subsequent liquid scintillation counting is described. This method may have application in the study of the genetically determined β-aminoisobutyric acid excretion in man.