An ESR (electron spin resonance) investigation of the effect of hydration water (D2O) on the absolute yields (G) of ion radicals in gamma-irradiated DNA at 77 K is reported. The total DNA radical yield is found to increase by over fourfold on addition of the primary hydration layer (20 water molecules/nucleotide) to DNA. At hydrations greater than ca. 20 waters/nucleotide the excess water freezes into a separate and apparently radiologically independent bulk ice phase. Ice formation steals ca. 5 waters from the hydration layer which causes a proportionate (by weight) drop in the total yield of DNA radical ions. Hydroxyl radicals (.OD) are not observed even at great signal amplification in the primary hydration layer but are found only in the ice phase, which suggests efficient transfer of holes and electrons from the hydration layer to the DNA; as a consequence, DNA and its associated hydration water form the target mass for radiation damage. These results show that water of hydration is critical to radical formation and stabilization in DNA; however, the ice surrounding DNA does not contribute to direct DNA damage and is found to have the same properties as bulk ice. At 18 waters per nucleotide (gamma = 18) about one-fourth of all ion radicals produced by the radiation are trapped at 77 K. The k value for destruction is found to vary only slightly with hydration. Based on the measured G and k values for hydrated DNA, a trapped radical ion cluster size is estimated to be about 6 nm. For hydrated DNA the cluster size suggests electron migration distances of about 17 bases (for migration along the DNA strand).