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.