The biogenesis of mitochondria requires the insertion of both nuclear and mitochondrially encoded proteins into the inner membrane. The inner membrane protein Oxa1 plays an important role in this process. Translocation of the terminal intermembrane space domains of subunit 2 of the cytochrome oxidase complex, Cox2, strictly depends on Oxa1. In contrast, other Oxa1 substrates can be inserted independently of Oxa1 function, although at reduced efficiency. A Saccharomyces cerevisiae mutant containing a large deletion in its mitochondrial genome allowed us to analyze the insertion process of a fusion protein of cytochrome b and Cox2. In this mutant, the N-terminal domain of Cox2 is synthesized as a hairpin loop that is flanked by hydrophobic transmembrane segments on both sides. Both genetic and biochemical evidences indicate that translocation of this region across the inner membrane still requires Oxa1 function. Thus, the position of intermembrane space domains within protein sequences does not appear to determine their dependence on the Oxa1 translocase. Our observations rather suggest that the dependence on Oxa1 correlates with the net charge of the domain that has to be translocated across the lipid bilayer.