We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable ␣-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a ␤-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitoninsolubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a ϳ400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.Most mitochondrial proteins are synthesized in the cytosol as preproteins, delivered to the mitochondrial surface by cytosolic factors such as hsp70 and mitochondrial import-stimulating factor, and transported to the intramitochondrial compartments by the preprotein import machinery of the outer and the inner membranes (the TOM 1 and TIM complexes, respectively)(1-4). The Saccharomyces cerevisiae TOM complex is composed of at least nine proteins (Tom71, -70, -40, -37, -22, -20, -7, -6, and -5) (5, 6). Tom40, the central component of the translocation channel, stably associates with the Tom22 receptor and small Tom components, Tom7, -6, and -5, and forms a ϳ400-kDa general insertion pore complex in yeast (5). Composition of the Neurospora crassa TOM complex is similar to that of S. cerevisiae, but Tom5 has yet to be identified in N. crassa. The mitochondrial inner membrane has two separate import machineries (7): the Tim23-Tim17 system and the Tim54-Tim22-Tim18 system. The Tim23-Tim17 system functions in the translocation of preproteins across the inner membrane in conjunction with Tim44, mhsp70, and GrpE (8 -10), whereas the Tim54-Tim22-Tim18 system functions in collaboration with the intermembrane space proteins Tim13, Tim12, Tim10, Tim9, and Tim8, in the import of proteins without a cleavable presequence, such as the phosphate carrier, the ADP/ATP carrier, and several Tim proteins (Tim23, Tim22, and Tim17) (10 -16).Although the fundamental mechanisms of mitochondrial protein import seem to be conserved from lower eukaryotes to mammals, only limited information is available for higher eukaryotic systems. Several mammalian counterparts have...