Conformational substates (CSs) of deoxyribonucleic acid (DNA), essential for its dynamics and function, are spectroscopically revealed by a new low-temperature method. Hydrated films of the sodium salt of DNA containing up to approximate to 100% of the B-form were quenched into the glassy metastable state at 77 K. Attainment of equilibrium in the glass --> liquid transition region by interconversion of CSs was followed isothermally by Fourier transform infrared spectroscopy. Kinetic analysis of difference spectra revealed two sequential relaxation processes and the spectral patterns of two distinct CSs of B-DNA, with exponential interconversion kinetics for each vibrational mode. The characteristic spectral features of the two CSs are also found in the spectrum of B-DNA in aqueous solution. The relevance of these CSs is discussed with respect to the crystalline state and DNA-protein interaction.