Cryo-electron microscopy and X-ray crystallography have shown that the pre- and postfusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C-3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C-3 symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C-3 axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C-3 symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. N-15 NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.