Previous studies showed that yeast VAS1 encodes both the cytoplasmic and mitochondrial forms of valyl-tRNA synthetase (ValRS), using alternative transcription and translation. The ValRS isoforms have identical polypeptide sequences, except for a 46-amino acid leader peptide that functions as a mitochondrial targeting signal. Although the two forms of the enzyme exhibit indistinguishable tRNA specificities in vitro, they cannot substitute for each other in vivo because of their different localizations. Here we show that the 46-residue leader sequence can be divided into two nonoverlapping peptides, each of which retains the ability to target the enzyme into mitochondria. The engineered proteins (with truncated leader sequences) are dual-targeted, rescuing both the cytoplasmic and mitochondrial defects of a vas1 knockout strain. Thus, in addition to alternative splicing and alternative translation initiation as mechanisms by which a single gene can encode cytoplasmic and mitochondrial activities, the inherent characteristics of a single polypeptide may enable it to be distributed simultaneously between two cellular compartments. This mechanism may explain how certain other single genes in Saccharomyces cerevisiae provide dual functions.