A combination of structures, energies, and spectral data calculated using density functional theory (DFT) with experimental NMR data has been used to assign conformational equilibria for tetracycline and 5a,6-anhydrotetracycline in water at pH 1, 7, and 10 and in chloroform (5a, 6-anhydrotetracycline) and methanol (tetracycline). The results suggest that tetracycline always prefers the extended conformation but that 5a, 6-anhydrotetracycline exists in water as a mixture of the two conformers and in chloroform exclusively in the twisted conformation. The conformational equilibria are also shown to be pH dependent.