The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5 phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal 5 P per dimer, 3 forms of the enzyme may exist in solution: apoenzyme 2 pyridoxal 5' phosphate, apoenzyme 1 pyridoxal 5' phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme 2 pyridoxal 5' phosphate/apoenzyme 1 pyridoxal 5' phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal 5 P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme 1 pyridoxal 5' phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4 pyridoxic 5 P (K(D) = 1.1 x 10-6M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by 2 independent methods, lend strong support to the hypothesis that the 2 subunits of the enzyme function independently.