Aqueous DNA solutions with or without the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) were exposed to gamma-rays at 77 K. After thawing the solutions, three experiments were carried out to confirm the generation of OH radicals in the hydration layer of DNA and to examine whether they act as an inducer of DNA strand breaks and base alterations. Observation with the ESR-spin trapping method showed ESR signals from PBN-OH adducts in the solution containing PBN and DNA, but there were few signals in the solution containing PBN alone, suggesting that reactive OH radicals were produced in the hydration layer of gamma-irradiated DNA and were effectively scavenged by PBN, and that unreactive OH radicals were produced in the free water layer of gamma-irradiated DNA. Agarose gel electrophoresis of DNA proved that PBN had no effect on the formation of strand breaks, whereas examination with the high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method showed that PBN suppressed the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). From these results it was concluded that OH radicals generated in the hydration layer of gamma-irradiated DNA did not induce DNA strand breaks but induced base alterations.