Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex. The TOM complex possesses two presequence binding sites on the cytosolic side (the cis site) and on the intermembrane space side (the trans site). Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to the cis and trans sites of the TOM complex. The N-terminal 14 residues of the presequence of subunit 9 of F(0)-ATPase are required for binding to the trans site. The interaction between the presequence and the cis site is not sufficient to anchor the precursor protein to the TOM complex. Tom7 constitutes or is close to the trans site and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import.