5-Diazouracil in monohydrated form showed mutagenicity and cytotoxicity on Salmonella typhimurium TA98 and TA100 strains without metabolic activation, and induced mouse micronucleated peripheral reticulocytes. Incubation of a plasmid supercoiled DNA with the compound caused DNA single-strand breaking: the supercoiled form was transformed into an open circular relaxed form and then into a linear form. The breaking was similarly caused in the absence of molecular oxygen. The breaking was not inhibited by superoxide dismutase and catalase, but inhibited by ethanol, butyl hydroxyanisole and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), suggesting the involvement of radical species other than oxygen-derived radical species. Sequencing analysis of the singly 5'-end-labeled DNA fragment showed that the phosphodiester breaking was not site-specific. When Escherichia coli cells were incubated with the compound, the intracellular double-strand DNA was fragmented. The fragmentation was inhibited by ethanol, DMPO, N-tert.-butyl-alpha-phenylnitrone (PBN) and thiol compounds. Generation of the carbon-centered radical was confirmed by the electron spin resonance spin-trapping technique using DMPO and PBN. The mutagenicity and the DNA breaking activity of 5-diazouracil can be ascribed to the carbon-centered radical.