Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner.