Cytoplasmically synthesized mitochondrial proteins are targeted to the organelle by an N-terminal presequence. We have identified a mutant ATP synthase beta-subunit with an altered presequence that results in a significant impairment of the transport of this polypeptide into mitochondria in vitro. When this mutant protein is expressed in yeast, its slow passage through the transport pathway interferes with the transport of a number of other mitochondrial proteins, indicating that they may share at least one common step in their transport into the mitochondrion.