Evidence for the roles of proteins and metal ions in the microheterogeneity of DNA damage is reviewed. Decondensation of chromatin in hypotonic buffers markedly sensitizes the DNA to radiation, while treatment of nuclei with hypertonic buffers strips the DNA of histones and other nuclear proteins and enhances the radiosensitivity of the DNA with respect to double-strand break (dsb) formation. Addition of the radical scavenger DMSO reduces the yield of strand breaks, but dehistonized chromatin remains approximately 2.5 times more sensitive to radiation than does native chromatin at 0.1 M DMSO. DNA-protein crosslink (DPC) formation is relatively unaffected by the removal of the majority of histones from chromatin. Most DPC form at or near the nuclear matrix, and matrix is stabilized and radiosensitized by Cu++. To elucidate the role of Cu++, the induction of dsb and DPC by gamma-radiation has been compared with that by hydroxyl radical from Fe(++)-EDTA, or Cu++ catalysed Fenton reactions. Data comparing the size of DNA fragments produced, the effect of expanding or dehistonizing chromatin, and the effects of radical scavengers suggest that gamma-radiation and Fe(++)-EDTA produce dsb at open chromatin sites, whereas Cu(++)-generated dsb are similar to radiation-induced DPC in their location at the nuclear matrix. Both metal ions appeared to produce damage by site-specific generation of hydroxyl radicals. The nuclear matrix, the proteinaceous skeleton which anchors chromosomal loops and provides sites for DNA replication and transcription, binds metal ions and matrix-attachment DNA regions (MARs) consisting of 300 + bp of AT-rich DNA. The interaction of cloned MARs with isolated nuclear matrices has been found to be hypersensitive to crosslinking upon gamma-irradiation, in comparison with associations formed by similarly sized DNA fragments lacking MAR sequences. Thus, the non-random distribution of radiation damage is partially explained by the protection of DNA afforded by histones and chromatin structure and partially by the hypersensitivity of DNA-nuclear matrix associations.