Apolipoprotein A-I (apoAI) contains several amphipathic α-helices. To carry out its function, it exchanges between lipid-free and different lipidated states as bound to membranes or to lipoprotein complexes of different morphology, size, and composition. When bound to membranes or to spherical lipoprotein surfaces, it is thought that most α-helices arrange with their long axis parallel to the membrane surface. However, we previously found that a central region spanning residues 87-112 is exclusively labeled by photoactivable reagents deeply located into the membrane (Córsico et al. (2001) J. Biol. Chem. 276, 16978-16985). A pair of amphipathic α-helical repeats with a particular charge distribution is predicted in this region. In order to study their insertion topology, three single tryptophan mutants, each one containing the tryptophan residue at a selected position in the hydrophobic face of the central Y-helices (W@93, W@104, and W@108), were used. From the accessibility to quenchers located at different membrane depths, distances from the bilayer center of 13.4, 10.5, and 15.7 Å were estimated for positions 93, 104, and 108, respectively. Reported data also indicate that distances between homologous positions (in particular for W@93 and W@104) are very short in dimers in aqueous solution, but they are larger in membrane-bound dimers. Data indicate that an intermolecular central Y-helix bundle would penetrate the membrane perpendicularly to the membrane surface. Intermolecular helix-helix interactions would occur through the hydrophilic helix faces in the membrane-bound bundle but through the hydrophobic faces in the case of dimers in solution.