The interactions of procainamide with DNA were studied by neutral and alkaline sucrose gradient sedimentation and sequential action of 2 enzymes: a mammalian repair endonuclease and bacterial DNA polymerase I. Sucrose gradient sedimentation shows that in the absence of photosensitization, the interaction of procainamide with DNA did not modify DNA sedimentation in alkaline or neutral sucrose gradients. In contrast, when a photosensitized DNA procainamide mixture was placed on sucrose gradients, the peak appearing on alkaline sucrose gradient after treatment with endonuclease was shifted toward the lower molecular weights, indicating that strand breaks had developed in the photosensitized procainamide DNA. Incubation of a [32P] labeled photosensitized procainamide-DNA complex with a repair endonuclease and DNA polymerase I released the label in the acid soluble fraction, indicating that only the photosensitized procainamide-DNA complex was susceptible to the endonucleolytic attack. There was only negligible release of the label in the acid soluble fraction without exposure of the DNA-procainamide mixture to light. The incorporation into DNA of [3H]-TTP (tritium labeled triphosphates) in presence of DNA polymerase I was inhibited when the photosensitized procainamide-DNA complex was used as substrate. However, after treatment of the photosensitized DNA complex with the repair endonuclease, the incorporation of [3H]-TTP was increased and reached values close to that observed with DNA unexposed to light, suggesting that the endonuclease functions as a repair enzyme.