Abstract A procedure, without prior fractionation of the subcellular components, has been developed for the simultaneous isolation of supernatant and mitochondrial aspartate transaminase (EC 2.6.1.1) from chicken heart or liver tissue homogenates. The procedure includes initial heat and ammonium sulfate fractionation. The two enzymes can then be separated by chromatography in hydroxylapatite. The supernatant enzyme elutes with 0.1 m potassium phosphate buffer, pH 6.9, and the mitochondrial enzyme with 0.3 m of the same buffer. The separated fractions can be further purified by ion exchange chromatography in carboxymethyl-Sephadex. The purified enzymes (isoenzymes) differ in amino acid composition and electrophoretic mobility. At pH 9.3 the supernatant enzyme is anionic and the mitochondrial is cationic. Antibodies against one isoenzyme fail to cross-react with the other protein in either Ouchterlony-type diffusion experiments or in the highly sensitive complement fixation test. Both enzymes contain pyridoxal phosphate as their prosthetic group at the active site which can be utilized to determine the substrate interactions with this site. The values for the substrate dissociation constants are: supernatant isoenzyme aspartate, 2 mm; oxalacetate, 35 µm; glutamate, 5 mm; α-ketoglutarate, 0.3 mm; mitochondrial isoenzyme aspartate, 0.6 mm; oxalacetate, 15 µm; glutamate, 11 mm; α-ketoglutarate, 1.2 mm. In addition, the values for the abortive complexes pyridoxal enzyme-keto acid are: oxalacetate, 62 µm, and α-ketoglutarate, 25 µm, for the supernatant enzyme, and oxalacetate, 50 µm, and α-ketoglutarate, 162 µm, for the mitochondrial transaminase. The absorption spectra of active site-bound pyridoxal phosphate at pH 8 differ in both isozymes, 355 nm maximal in the mitochondrial and 365 nm in the supernatant enzyme. The isozymes are present in chicken liver and heart. Amino acid composition, spectral properties, electrophoretic migration, and immunological reactivity, established identity among any given isozyme of the same cytological localization independent of tissue origin. Both the supernatant and mitochondrial aspartate transaminases contain multiple forms which are electrophoretically detectable. All the subforms possess catalytic activity.