Transport of preproteins across the inner mitochondrial membrane requires the action of the matrix heat shock protein Hsp70. Together with its co-chaperone mitochondrial GrpE (Mge1), mtHsp70 transiently binds to the inner membrane translocase subunit Tim44 in a nucleotide-regulated manner, forming an ATP-dependent import driving machinery. We report that a mutant form of Mge1 (Mge1-100) is completely absent in mtHsp70-Tim44 complexes, although its ability to interact with soluble mtHsp70 is only partially reduced. While this mge1-100 mutation only partially retards preprotein translocation into the matrix, it exerts a selective effect on sorting of cytochrome b 2 to the intermembrane space. A cytochrome b 2 with an altered sorting signal, which is only processed to the intermediate stage and mistargeted to the matrix of wild-type mitochondria, is processed to the mature form and correctly targeted to the intermembrane space of mge1-100 mitochondria. These results suggest that (1) Mge1-100 discriminates between soluble and membrane-bound mtHsp70 and (2) the membrane-bound mtHsp70-Mge1 driving system competes with the sorting machinery for translocation of preproteins like cytochrome b 2 .