In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX% gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5u so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.