Strand breaks can be produced in pBluescript plasmid DNA by S-nitrosothiol and H2O2 in the presence of a metal chelator, diethylenetriaminepentaacetic acid. S-Nitrosothiols with a wide range of stability were found to be active as a DNA cleaver in the presence of H2O2. Strand breaks were temperature dependent, occurring more rapidly at higher temperature. Sodium azide and mannitol inhibited S-nitrosothiol/H2O2-induced strand breaks in DNA. Catalase inhibited damage to DNA in a concentration-dependent manner whereas both superoxide dismutase and a yeast protector protein did not prevent damage to DNA. It is suggested that observed strand breaks in DNA are mediated by hydroxyl radicals arising from the reaction between H2O2 and thiyl radicals generated by homolytic decomposition of S-nitrosothiol.