Polarized attenuated total reflectance (ATR), grazing angle reflection-absorption, and transmission Fourier transform infrared (FT-IR) spectroscopy methods have been used to investigate the orientation of peptide nanotubes in the functionally relevant environment of ordered phospholipid multibilayers. Eight-residue cyclic peptides of alternating D- and L-amino acids which self-assemble to form open-ended, tubular structures have been shown previously to function as transmembrane channels for ion transport. Although the tubular structure of the peptide assembly has been established in the pure solid state, this study presents the first detailed biophysical investigation of the peptide nanotubes within the context of the lipid film to afford a quantitative estimate of their angle of orientation relative to the lipid bilayer. We find by ATR IR that in accordance with the previous structure-function model hypothesis for a transport-competent channel, the central axis of nanotubes composed of cyclo[(L-Trp-D-Leu)(3)-L-Gln-D-Leu] is aligned parallel to the dimyristoyl phosphatidylcholine (DMPC) hydrocarbon chains, at approximately 7 degrees from the axis normal to the bilayer plane. This upright orientation for the DMPC/peptide film is also qualitatively supported by grazing angle and transmission FT-IR data. By contrast, FT-IR, studies of two- and three-dimensionally ordered peptides, Langmuir-Blodgett films and microcrystals, respectively, show that the peptide tubes lie parallel to the substrate surface in the absence of a hydrophobic supportive environment. These studies support a transport-competent, membrane-spanning orientation of the peptide nanotubes in the lipid bilayers. Furthermore, they indicate that the orientation of the tubes can be controlled by a supporting environment, implicating biological and nanotechnological applications.