The TIM23 complex of the mitochondrial inner membrane mediates the import of preproteins that contain positively charged targeting signals. This translocase consists of the two phylogenetically related membrane-embedded subunits Tim17 and Tim23 to which four largely hydrophilic subunits, Tim50, Tim44, Tim16, and Tim14, are attached. Whereas in vitro reconstitution experiments have suggested a pore-forming capacity of recombinant Tim23, virtually nothing is known about the properties and function of Tim17. We employed a combined genetic and biochemical approach to address the function of Tim17 in preprotein translocation. Tim17 exposes an N-terminal hydrophilic stretch into the intermembrane space. Truncation of the first 11 amino acid residues of this stretch did not affect the stability or integrity of TIM23 subunits but strongly impaired the import of preproteins. Moreover, expression of the truncated Tim17 variant led to a dominant negative effect on the mitochondrial membrane potential. By an alanine-scanning approach we identified two conserved negative charges in the N terminus of Tim17 as critical for Tim17 function. The replacement of these positions by positively charged residues results in a strong growth defect, which can be cured by reverting two conserved positive charges into aspartate residues between transmembrane domains two and three of Tim17. On the basis of these observations we propose that charged residues in Tim17 are critical for the preprotein-induced gating of the TIM23 translocase.