Ethidium causes the cleavage of DNA via a light and oxygen dependent process. Using covalently closed circular DNA as a substrate, the saturation kinetics and the dependence on superhelical density of the cleavage indicate that intercalated ethidium is mainly responsible for nicking DNA. Superoxide dismutase has little effect on the reaction and catalase none. Lowering the pH inhibited the reaction. The reaction mechanism and its use in determining superhelical densities of covalently closed circular DNA's are discussed.