The role of histones and higher order chromatin structures in protecting against oxidative DNA damage was investigated using an in vitro system consisting of nuclear and nucleoid monolayers as model chromatin substrates. These substrates, derived from human skin fibroblasts, were challenged with hydroxyl radicals produced via a Fenton reaction involving Fe(ll)-ethylenediaminetetraacetic acid and ascorbic acid. The resulting DNA strand breaks were measured using the alkaline unwinding technique. The sequential removal of chromosomal proteins from the DNA by pretreating nuclear monolayers with increasing concentrations of salt dramatically increased the frequency of hydroxyl radical-induced DNA strand breaks. Furthermore, the DNA in decondensed chromatin was found to contain 14-fold fewer DNA strand breaks than naked, supercoiled DNA, whereas the DNA of “native” chromatin and “condensed” chromatin contained 100-fold and 300-fold fewer breaks, respectively. We conclude that the binding of histones to the DNA and its organization into higher order chromatin structures dramatically protects the DNA against hydroxyl radical-induced DNA strand breaks and thus should be considered part of the cellular defense against the induction of oxidative DNA damage. © 1992 Wiley-Liss, Inc.