A rapid method for purification of mitochondrial aspartate aminotransferase from rat liver employing high-performance liquid chromatography is reported. The product is purified 80-fold with a recovery greater than or equal to 50% in a single day. The amino acid composition, N-terminal amino acid sequence, specific activity, and spectral characteristics of the isolated enzyme are similar to those previously reported for this protein. The protein is homogeneous by standard electrophoretic and chromatographic criteria, but can be resolved into at least five isoforms by a carboxymethylated resin column using high-performance liquid chromatography. The principal isoform initially isolated is converted into two additional isoforms with lower specific activity upon storage at 4 degrees C.