The mechanism by which L-histidine modulates H2O2-induced damage to DNA has been investigated by alkaline and neutral gel electrophoresis of cellular DNA, by measuring the conversion of purified supercoiled DNA to its relaxed and linear forms and by the ESR spin-trapping technique. L-Histidine greatly increased the amount of H2O2-mediated DNA single-strand breaks. DNA double-strand breaks were produced only in cells exposed to H2O2 and L-histidine. The addition of a cell permeable chelator such as o-phenanthroline (unlike EDTA, DTPA and desferrioxamine) prevented both DNA single- and double-strand breakage induced by H2O2 plus L-histidine. In vitro, the profile of the dose-response curve for the ferrous iron-mediated, H2O2-dependent DNA nicking was modified by the addition of L-histidine. At low H2O2 concentrations, corresponding to the maximum extent of DNA cleavage, L-histidine was protective. At higher H2O2 concentrations L-histidine enhanced the formation of DNA single-stand breaks and produced DNA double-strand breaks. The increase in H2O2-mediated DNA nicking by L-histidine depended on the L-histidine:Fe(II) ratio, the maximal rate occurring at a molar ratio of 10(3):1 and being independent of the concentration of DNA. Thus, it appeared that intracellular iron mediated both DNA single- and double-strand breaks induced by H2O2 plus L-histidine. Results of ESR experiments seemed to rule out the involvement of the hydroxyl radical by itself in DNA cleavage mediated by the L-histidine:Fe(II):H2O2 system.