We have examined the effect of Trp and Phe residues on the positioning of a poly-Leu transmembrane helix relative to the microsomal membrane by employing a previously described "glycosylation mapping" technique [Nilsson, I. M., Sääf, A., Whitley, P., Gafvelin, G., Waller, C., and von Heijne, G. (1998) J. Mol. Biol. 284, 1165-1175]. Both Trp and Phe tend to push the transmembrane helix into the membrane when inserted in positions flanking the poly-Leu stretch, and Trp (but not Phe) pulls the transmembrane helix toward the lipid-water interface when inserted inside the poly-Leu segment. Thus, the preference of Trp for the lipid-water interface previously suggested on the basis of biophysical studies of model peptides can also be observed for a bona fide transmembrane helix in a biological membrane. We further show that a sufficiently long poly-Trp segment functions as an efficient stop-transfer sequence during protein translocation across the microsomal membrane, despite the preference of Trp residues for the lipid-water interface region.