TIMM50 is a component of the yeast Tim23 import machinery, which mediates translocation of presequence containing proteins across the mitochondrial inner membrane. By searching sequence databases, open reading frames encoding proteins with similarity to Tim50 and with a putative mitochondrial presequence were identified in the genomes of evolutionarily distant organisms, including human. Tim50 was found to interact with the N terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti Tim50 antibodies blocked the protein translocation across the inner membrane. A translocation intermediate accumulated at the translocator of the outer mitochondrial membrane (TOM) complex was cross linked to Tim50. Thus it can be concluded that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the Tim23 complex.