Mitochondria, the site of cellular energy production, must import all proteins necessary for their function. Import is mediated by two mechanisms: the translocase of the outer membrane (Tom) and the translocase of the inner membrane (Tim). Tim23 and Tim17 are integral membrane proteins that associate to form the import channel for mitochondrial preproteins that contain N-terminal hydrophilic sequences. They also associate with Tim44, an adaptor for the membrane binding of mtHsp70, a matrix heat shock protein, which drives the import of the processed preprotein. The N-terminal intermembrane space domain of Tim23 contains a leucine zipper motif and mediates the formation of a Tim23 dimer. As an imported protein passes through the TOM machinery, its N-terminal matrix targeting sequence interacts with the Tim23 dimer. This induces the dissociation of the dimer and initiation of inner membrane translocation of the presequence. In addition to its 9 kDa N-terminal hydrophilic segment, Tim23 contains a 14 kDa hydrophobic domain with four predicted membrane spans. Thus, Tim23 is an important integral membrane component of the mitochondrial protein translocation machinery.