D-3-Aminoisobutyrate-pyruvate aminotransferase was purified 2000-fold from rat liver extract using heat treatment, ammonium sulfate fractionation, carboxylmethyl-Sepharose CL-6B, DEAE-Sepharose CL-6B, hydroxyapatite, Sephacryl S-200 and electrofocusing chromatographies. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 220 kDa and the subunit molecular mass was 52 kDa. The enzyme exhibited absorption maxima at 280 nm and 412 nm with a shoulder at 330 nm at neutral pH. The pH optimum for enzyme activity was 9.5 and the Km values for beta-alanine and pyruvic acid were calculated to be 0.81 mM and 0.45 mM, respectively. The purified enzyme catalyzed the transamination of omega-amino acids; beta-alanine and D-3-aminoisobutyric acid served as good amino donors, and pyruvic acid, glyoxylic acid and oxaloacetic acid were favorable amino acceptors. 6-Azauracil and 6-azathymine were found to be potent inhibitors of purified rat liver D-3-aminoisobutyrate-pyruvate aminotransferase. 6-Azauracil acted as a competitive inhibitor with respect to beta-alanine, and was an uncompetitive inhibitor with respect to pyruvic acid with a Ki of approximately 8.9 mM.