The low-temperature dynamics in nonoriented hydrated films of the sodium salt of the d(CGCGAATTCGCG)2 dodecamer with either 13 (14) or 8 water molecules per nucleotide (Gamma = 13 (14) or 8) was studied by Fourier transform infrared (FTIR) spectroscopy. This dodecamer is ideal for this study because it remains in the B form even at low water activity, and thus, we do not have to worry about IR bands from the A form. The effects of cooling rate and of isothermal relaxation at 180 and 200 K on B-DNA's conformer substates, B-I and B-II, were revealed by a combination of IR difference spectroscopy and by determining B-II/B-I population ratios via careful curve resolution of IR spectra. On slow cooling a dodecamer film with Gamma = 13 (14) from 290 to 180 K at a rate of 2 K min(-1), B-II transforms into B-I fast enough to remain equilibrated down to 180 K. However, on fast-cooling of the same sample at a rate of approximate to60 (or 90) K min(-1), a nonequilibrium state is frozen-in. Unexpectedly, on isothermal relaxation at 180 or 200 K toward equilibrium, B-I converts into B-II; that is, the B-II/B-I equilibrium line is approached from below. This isothermal B-I --> B-II transition is coupled with restructuring of their hydration shells such that increased hydrogen-bond interaction stabilizes B-II. A dodecamer film with Gamma = 8 behaves very differently: the B-II/B-I population ratio remains equilibrated both on slow-cooling and on fast-cooling to 180 K, and thus, isothermal relaxation does not occur. Therefore, the falling-out of equilibrium on fast-cooling of the dodecamer with Gamma = 13 (14) must be caused by one or more of these additional water molecules. We conclude that, with these additional water molecules as part of the hydration shells, restructuring of the B-I and B-II hydration shells is a comparably slow process, whereas B-II --> B-I is much faster. In this scenario, first restructuring of the hydration shells slows down on fast-cooling, and the B-II/B-I equilibrium line is left at approximate to240-220 K. However, the B-II --> B-I transition continues until a B-II/B-I ratio is frozen-in which is below that of the B-II/B-I equilibrium ratio. Because the frozen-in hydration shells cannot stabilize B-II anymore by additional hydrogen bonding, the B-I state is favored in the frozen-in nonequilibrated state. We further show that the less complete IR-spectroscopic analysis of a native polymeric DNA with Gamma = 13 reveals the same effects as the dodecamer and, thus, that our conclusions seem to be general and not restricted to the synthetic oligonucleotide. Isobestic points in the IR spectra of the dodecamer and of native polymeric B-DNA recorded on isothermal relaxation at 200 K indicate that only two species are involved. Thus, the low-temperature dynamics of B-DNA seems to be caused solely by the processes described above. Our findings enable us to interpret the inelastic neutron scattering spectroscopy analysis reported by Sokolov et al. (J. Chem. Phys. 1999, 110, 7053-7057), and they support their speculation that the glassy dynamics in DNA is ruled by water of hydration. We further speculate that slowing down of restructuring of the B-I and B-II hydration shells at approximate to220-240 K is the cause for the suppression of the biological functions at low temperatures.