We present isotherm and X-ray reflectivity (XR) measurements from Langmuir monolayers of a de novo synthetic di-alpha -helical peptide; consisting of two identical 31-residue, mostly alpha -helical peptide units joined by a disulfide bond at their amino-termini. Fitting the XR data to slab models shows that the dihelices lie in the plane of the interface at low pressures. The monolayers were insufficiently stable for study at high pressures, but Langmuir films based on a derivative of the peptide alkylated at its amino termini permitted investigations over a larger range of pressures. We observed an orientational transition, in which the alpha -helices begin by lying in the plane of the interface at low surface pressures and orient themselves approximately normal to the interface at high pressures. We draw the same conclusions from the XR data when we analyze it using box refinement, an iterative, model-independent method for recovering structure from XR data. Mixtures of these palmitoylated peptides with a fatty acid (palmitic acid) or a phospholipid (DLPE) behaved similarly. None of the systems produced peaks in the grazing incidence diffraction signal indicative of long-range ordering of the upright a-helices. Off-specular in-plane scattering measurements based on the difference signal between the peptide/DLPE mixture and pure DLPE suggest that the peptide achieves only liquidlike order within the plane. We discuss the implications and prospects for future work on designed peptide monolayers incorporating prosthetic groups that could be used to study electron transfer in proteins and provide a basis for biomolecular electronics applications.