Several pharmacologically important barbituric acids were examined by proton magnetic resonance spectroscopy in trifluoroacetic acid, p-dioxane, and dimethyl sulfoxide-d6. The data obtained greatly facilitate identifying and distinguishing barbiturates. Variations in chemical shift with alkyl substitution have been discussed, and the suggestion has been put forward that—the CH3(Et) protons may be a sensitive probe for detecting changes in anisotropy (both ring and C=O) in a series of barbiturates. Evidence for the preferred configuration of C-5 phenyl, ethyl, and isopropyl substituents was obtained. Concentration studies by both proton magnetic resonance and infrared spectroscopy indicate that the barbiturates examined exist predominantly as solvated monomers rather than as dimers or oligomers.