The liquid crystalline alignment medium based on a quasi-ternary surfactant system comprising cetylpyridinium bromide (CPBr)/hexanol/sodium bromide [6.5% CPBr/hexanol (1/1.33, w/w), 30 mM NaBr in 10 mM glycine/HBr buffer, pH 3.0] was characterized. We assessed the morphology of the amphiphilic aggregates by electron microscopy. The images show a network of elongated wormlike structures that are 5.5 nm wide ranging up to 1 mum in length. In the bulk medium, they are closely packed with an average center-to-center spacing of similar to5 nm as measured by negative staining and similar to10 nm as determined directly by cryo-electron microscopy. Solid-state H-2 and P-31 NMR spectroscopies were used to determine the orientational order parameters of the aromatic ring and the aliphatic chain of the surfactant as well as the alcohol. Order parameter analysis revealed that the pyridinium ring is tilted with respect to the CPBr molecular symmetry axis. The cetyl chain has the typical order profile of hydrocarbon chains in liquid-crystalline phases: an order plateau for methylene segments near the pyridinium ring and rapidly decreasing order for the second half of the chain toward the terminal methyl group. Hexanol shows a similar order profile. However, its order is much lower than the order of the CPBr chains. To demonstrate the efficacy of this alignment medium for structure determination by NMR, we measured a large set of residual dipolar couplings for the potent HIV-1 inactivating protein, cyanovirin-N, determined the orientation of the alignment tensor, and verified the fold of this molecule at low pH.