Signal sequences for insertion of protein into the mammalian endoplasmic reticulum orient themselves in the translocon on the basis of their flanking charges. It has recently been shown that hydrophobic N-terminal signals initially insert head-on before they invert their orientation to translocate the C-terminus. The rate of inversion is reduced with the increasing hydrophobicity of the signal due to an increased affinity for the initial bound state at the translocon. To probe the environment of the signal while its orientation is determined, different hydrophobic residues were inserted at various positions throughout a uniform oligoleucine signal sequence and the constructs were expressed in transfected COS-7 cells. The resulting topologies revealed a strikingly symmetric position dependence specifically for bulky aromatic amino acids, reflecting the structure of a lipid bilayer. Maximal N-translocation was observed when the guest residues were placed at the N- or C-terminus of the hydrophobic sequence or in the very center, corresponding to the positions of highest expected affinity of the signal sequence as a membrane-spanning helix for the bilayer. The results support the model that during topogenesis in vivo the signal sequence is exposed to the lipid membrane.