Clustered damage induced by ionizing radiation--two or more oxidized bases, abasic sites, or strand breaks within a few DNA helical turns--have been postulated to be major lethal and/or mutagenic sites. Although they have recently been shown to be induced in genomic DNAs by ionizing photons and particles, little is known of the factors that affect their yields or the relative levels of the classes of clusters. Toward this aim we have investigated the effect of DNA milieu, specifically, a nonradioquenching (phosphate) or radioquenching (Tris) solution, upon the generation of clustered lesions in a well-defined molecule, T7 bacteriophage DNA. Irradiation of DNA in Tris reduces the yields of all clustered damages to 1-3% of the levels formed in phosphate. Further, although the percentage of the total clusters in oxidized purine clusters is largely unchanged, and the level of abasic clusters decreases, the frequencies of double-strand breaks and oxidized pyrimidine clusters increase in the radioquenching solution. The ratio of the level of oxidized pyrimidine clusters to double-strand breaks in a DNA in radioquenching solution is similar to that obtained in DNA in human cells, also a radioquenching environment.