Nonoriented hydrated films of the sodium salt of the d(CGCGAATTCGCG)(2) dodecamer, with Gamma (water molecules per nucleotide) = 20, 14, 8, and 6, were investigated by Fourier transform infrared spectroscopy. Curve resolution of the spectral region containing the symmetric stretching vibration of the ionic phosphate at two temperatures allows estimation of B-DNA's B-I/B-II conformer substate population ratio, The B-I/B-II population ratio decreases at 290 K with decreasing water activity from 1.4 for Gamma = 20 to 1.2, 1.0, and 0.72 for Gamma = 14, 8, and 6. Increasing B-II population with decreasing water activity is attributed to differences in hydration as the driving force for the B-I --> B-II transition. An enhanced B-II population could be significant for the interaction of proteins with B-DNA in chromatin. We surmize that the B-II substate could be an intermediate in the transition of canonical B- to A-DNA.