Abstract: The import of precursor proteins into mitochondria represents a cell biological process that is absolutely required for the survival of an eukaryotic cell. A complex chain of reactions needs to be followed to achieve a successful transport of mitochondrial proteins from the cytosol through the double membrane system to their final destination. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Although most of the protein components of the import machinery have been identified and functionally characterized using this basic system, the definition of the molecular mechanisms requires more specialized assay techniques. Here we describe modifications of the standard in vitro import assay technique that are based on the utilization of recombinant preprotein constructs. The application of saturating amounts of substrate preproteins is a prerequisite for the determination of translocation kinetics and energy requirements of the import process. Accumulation of preproteins as membrane-spanning translocation intermediates further provides a basis for the functional and structural characterization of the active translocation machinery.