The rat and human mitochondrial branched chain aminotransferase (BCAT(m)) cDNAs have been isolated and shown to encode mature proteins of 41.2 and 41.3 kDa with presequences of 27 amino acids. When rat BCAT(m) is overexpressed in COS-1 cells, the protein exhibits BCAT activity and correct processing of the mitochondrial targeting sequence. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human BCAT(m) gene resides on chromosome 19 and the human cytosolic enzyme (BCAT(c)) gene on chromosome 12. Finally, the nomenclature BCAT1 for the cytosolic gene and BCAT2 for the mitochondrial BCAT gene is proposed.