Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH2 nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH2-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.