Dual localization of proteins in the cell has appeared in recent years to be a more abundant phenomenon than previously reported. One of the mechanisms by which a single translation product is distributed between two compartments, involves retrograde movement of a subset of processed molecules back through the organelle-membrane. Here, we investigated the specific contribution of the mitochondrial targeting sequence (MTS), as a cis element, in the distribution of two proteins, aconitase and fumarase. Whereas the cytosolic presence of fumarase is obvious, the cytosolic amount of aconitase is minute. Therefore, we created (1) MTS-exchange mutants, exchanging the MTS of aconitase and fumarase with each other as well as with those of other proteins and, (2) a set of single mutations, limited to the MTS of these proteins. Distribution of both proteins is affected by mutations, a fact particularly evident for aconitase, which displays extraordinary amounts of processed protein in the cytosol. Thus, we show for the first time, that the MTS has an additional role beyond targeting: it determines the level of retrograde movement of proteins back into the cytosol. Our results suggest that the translocation rate and folding of proteins during import into mitochondria determines the extent to which molecules are withdrawn back into the cytosol.