Phenylalanyl-tRNA synthetases from mitochondria of yeast and hen liver resemble their corresponding cytoplasmic counterparts. Whereas slight intraspecies differences at the amino acid binding site, reflecting variations in the structures of these distinct enzymes, are exploitable by phenylalanine analogues, no intraspecies difference can be noted for the strategies to achieve the high fidelity of protein synthesis. While the yeast mitochondrial enzyme follows the pathway of posttransfer proofreading, the hen liver mitochondrial enzyme uses a tRNA-dependent pretransfer proofreading in the case of the natural amino acids. The accuracy of mitochondrial phenylalanyl-tRNA synthetases appears to be even better than the accuracy of the corresponding cytoplasmic enzymes. Interspecies rather than intraspecies differences for the functional role of certain amino acid residues of the enzymes further indicate the close relationship of the intracellular heterotopic isoenzymes. By use of a highly sensitive immunospotting procedure, common antigenic determinants are detected only within the enzymes from the two intracellular compartments of the same organism. The results suggest the origin of the cytoplasm-mitochondrion isoenzyme pair by independent gene duplication of the ancestral nuclear gene. A similarity of mitochondrial enzymes to the phenylalanyl-tRNA synthetase from Escherichia coli is not observed.