The mitochondrial isoenzyme of aspartate amino-transferase (mAspAT; subunit Mr 45,000) is synthesized on free polysomes in the cytosol as a precursor of higher Mr (pre-mAspAT; Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). We have isolated three overlapping cDNA clones that correspond almost to the full length of pre-mAspAT mRNA with 100 nucleotides at the 5' end missing. The mRNA is 2.1 kilobase pairs long and has a 3' noncoding region of 0.7 kilobase pairs. The cDNAs code for the 401 amino acid residues of mAspAT plus an NH2-terminal pre-piece. Deviations from the reported amino acid sequence were found at positions 154 and 202 where the cDNA specifies Gln instead of Glu. The pre-piece consists of 22 amino acid residues, among them 4 arginine and no acidic residues.