Import of proteins into mitochondria is a pivotal process in the biogenesis of mitochondria. Only about 1% of the 1000-2000 different proteins constituting the mitochondrion are encoded in the mitochondrial deoxyribonucleic acid (DNA). All others are specified by nuclear genes. They are translated in the cytosol and released from ribosomes as precursor proteins, which are then translocated into the various mitochondrial subcompartments. In the past, a variety of methods has been developed to study the process of import. An important tool is the use of in vitro import systems using isolated mitochondria and precursor proteins synthesized in cell-free systems. Together with the use of genetic and biochemical methods, this led to the identification of several translocation machineries consisting of a large number of components.