Mitochondria produce essentially all of the energy for tissues with high metabolic activity, such as heart and brain. Proper targeting and import of precursor proteins into mitochondria is crucial to their function. Currently, precursor proteins targeted to mitochondria are thought to be translated in the cytosol, targeted to the mitochondria by their transit peptide, and imported post-translationally via a multisubunit import complex in the mitochondrial membrane. However, we have found that, in addition to receptors in the outer mitochondrial membrane (OM) which recognize these precursor proteins, novel receptor(s) for the translation machinery also exist in the OM. Using three independent methods based on spectroscopic, SDS-PAGE, and radiometric assays, we have shown that ribosomes programmed with nascent mitochondrial precursor proteins specifically bind to the OM in a reaction controlled by both GTP and the transit peptide of the precursor protein. In addition, we also report the novel finding of a group of GTP-binding proteins in the mitochondrial membrane,one of which, termed G54, interacts with ribosomes to hydrolyze GTP. GTP will specifically cause the release of non-programmed ribosomes, or ribosomes programmed with non-mitochondrial proteins, from the OM but does not cause release of ribosomes programmed with mitochondrial precursor proteins. Ribosomes programmed with mitochondrial proteins also stably bind in the presence of GDP but the non-hydrolyzable GTP-analogue, guanosine-5'-[thio]-triphosphate, severely attenuates binding. These data show that, 1) Novel receptors specific for ribosomes are present on the mitochondrial OM, 2) Ribosome binding is mediated by both a GTP-dependent process, and the nature of the nascent peptide chain, and 3) A family of GTP-binding proteins is present in the mitochondrial membrane of which one, G54, participates in ribosome binding. These findings strongly support the controversial, but logical, hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria. The significance of these findings is that co-translational import may represent the major protein import pathway into mitochondria opening new directions and reagents to explore both normal, and diseased, metabolism in tissues with high energy needs, such as heart and brain.