The hydration of six B-DNA dodecamers with A-tracts of variable length and sequence has been investigated via the nuclear magnetic relaxation dispersion (NMRD) of the water H-2 and O-17 resonances. By subdividing the aqueous DNA solution into microscopic emulsion droplets, NMRD measurements could be performed at -20 degrees C, thereby greatly enhancing the sensitivity of the method. The NMRD profiles show that all six dodecamers contain long-lived water molecules. These water molecules are displaced by netropsin and must therefore be located in the minor groove. The number of long-lived water molecules is correlated with the width of the minor groove as seen in crystal structures. The NMRD data are consistent with a single file of 3-9 long-lived water molecules located at the base pair steps and extending 1-2 steps on either side of the A-tract. Dodecamers with central A-tracts of sequence A(4)T(4), T(4)A(4), and (AT)(4) are found to contain seven or nine long-lived water molecules, challenging the common view that T-A steps widen the minor groove and disrupt the hydration structure. The long-lived water molecules observed here are highly ordered with an entropy comparable to that of water molecules in ice, but most of them undergo a symmetric flip motion while residing in the groove. The mean water residence time is essentially the same, 10-15 ns at -20 degrees C, for all investigated dodecamers, suggesting that water exchange occurs from an open state with a uniformly wide minor groove. From the temperature dependence of the water residence time, an activation enthalpy of 53 kJ mol(-1) is obtained for this process.