The critical residues involved in targeting and processing of the soybean alternative oxidase to plant and animal mitochondria was investigated. Import of various site-directed mutants into soybean mitochondria indicated that positive residues throughout the length of the presequence were important for import, not just those in the predicted region of amphiphilicity. The position of the positive residues in the C-terminal end of the presequence was also important for import. Processing assays of the various constructs with purified spinach mitochondrial processing peptidase showed that all the -2-position mutants had a drastic effect on processing. In contrast to the import assay, the position of the positive residue could be changed for processing. Deletion mutants confirmed the site-directed mutagenesis data in that an amphiphilic alpha-helix was not the only determinant of mitochondrial import in this homologous plant system. Import of these constructs into rat liver mitochondria indicated that the degree of inhibition differed and that the predicted region of amphiphilic alpha-helix was more important with rat liver mitochondria. Processing with a rat liver matrix fraction showed little inhibition. These results are discussed with respect to targeting specificity in plant cells and highlight the need to carry out homologous studies and define the targeting requirements to plant mitochondria.