Both the precursor and the mature form of mitochondrial aspartate aminotransferase were synthesized in a cell-free coupled transcription/translation system directed by the recombinant expression plasmid pOTS-pmAspAT and pOTS-mAspAT, respectively. Both newly synthesized forms of the protein were imported into isolated mitochondria, with the precursor correctly processed to the mature form. In both cases the import process showed resistance to externally added pronase and was abolished in mitochondria treated with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Moreover the imported products showed the same intramitochondrial localization as judged by a subfractionation procedure. In both cases import was time dependent and was completed in about 15 min. Finally a competitive inhibition of the import of the precursor of aspartate aminotransferase was found due to externally added purified aspartate aminotransferase.