A soluble aminotransferase with kynurenine aminotransferase (KAT) activity has been recently isolated from rat brain. This enzyme corresponds to a cytosolic form of glutamine transaminase K (GTK). In addition to the cytosolic enzyme, a mitochondrial-associated form of this KAT/GTK also exists. In the present work we have isolated a rat brain cDNA clone encoding a KAT/GTK enzyme identical to the soluble form but carrying an additional stretch of 32 amino acids at its NH2-terminus. Several structural features of this sequence resemble those of leader peptides for mitochondrial import. Evidence that the isolated cDNA encoded for mitochondrial KAT/GTK was obtained after transfection of HEK-293 cells with the cDNA coding for this new KAT/GTK isoenzyme. In fact, a significant enrichment of both KAT and GTK enzymatic activities was found in the crude mitochondrial fraction of the transfected cells.