The requirements for protein import into mitochondria was investigated by using the targeting signal of the F(A)d subunit of soybean mitochondrial ATP synthase attached to two different passenger proteins, its native passenger and soybean alternative oxidase. Both passenger proteins are soybean mitochondrial proteins. Changing hydrophobic residues at positions -24:25 (Phe:Leu), -18:19 (Ile:Leu) and -12:13 (Leu:Ile) of the 31 amino acid cleavable presequence gave more than 50% inhibition of import with both passenger proteins. Some other residues in the targeting signal played a more significant role in targeting of one passenger protein compared to another. Notably changing positive residues (Arg, Lys) had a greater inhibitory affect on import with the native passenger protein, i.e. greater inhibition of import with F(A)d mature protein was observed compared to when alternative oxidase was the mature protein. When using chimeric passenger proteins it was shown that the nature of the mature protein can greatly affect the targeting properties of the presequence. In vivo investigations of the targeting presequence indicated that the presequence of 31 amino acids could not support import of GFP as a passenger protein. However, fusion of the full-length F(A)d coding sequence to GFP did result in mitochondrial localisation of GFP. Using the latter fusion we confirmed the critical role of hydrophobic residues at positions -24:25 and -18:19. These results support the proposal that core mitochondrial targeting features exist in all presequences, but that additional features exist. These features may not be evident with all passenger proteins.