The cleavable pre-sequences of imported chloroplast and mitochondrial proteins have several features in common. This structural similarity prompted us to test whether a chloroplast pre-sequence (;transit peptide') can also be decoded by the mitochondrial import machinery. In the green alga, Chlamydomonas reinhardtii, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (a chloroplast protein) is nuclear-encoded and synthesized in the cytosol with a transient pre-sequence of 45 residues. The 31 amino-terminal residues of this chloroplast pre-sequence were fused to mouse dihydrofolate reductase (a cytosolic protein) and to yeast cytochrome oxidase subunit IV (an imported mitochondrial protein) from which the authentic pre-sequence had been removed. The chloroplast pre-sequence transported both attached proteins into the yeast mitochondrial matrix or inner membrane, although it functioned less efficiently than an authentic mitochondrial pre-sequence. We conclude that mitochondrial and chloroplast pre-sequences perform their function by a similar mechanism.