Isolation on a preparative scale of crystalline pyridoxal phosphate-dependent threonine dehydratase (responsible for threonine deamination) from rat liver tissue is described. The enzyme was purified by stepwise salting out with (NH4)2SO4, two precipitations with acetone, gel filtration through Sephadex G-25, chromatography on DEAE cellulose, repricipitation with ammonium sulfate and crystallization. The ratio of threonine to serine dehydratase activities was altered only slightly through all the steps of the purification procedure. Both enzymes proved to be similar in their chromatographic properties; this suggests that a single enzyme is responsible for dehydrative deamination of both hydroxyamino acids in rat liver tissue. Stability of the enzyme preparations was distinctly increased after DEAE cellulose chromatography. The yield of crystalline threonine (serine) dehydratase was about 3%; the enzyme was purified 1500-1800-fold.