Lignin biopolymers constitute 30% of plant biomass and belong to the most abundant organic polymers on earth. We showed previously that this important component of dietary fiber exhibited a protective effect against the overall DNA damage induced by H2O2 or N-methyl-N'-nitro-N-nitrosoguanidine in hamster lung cells and human foreskin cells cultured in vitro. The objective of the present work was to examine DNA-protective effects of lignin in rat testicular cells and rat peripheral blood lymphocytes using in vitro and ex vivo experiments. H2O2 and visible light-excited methylene blue (MB) were used as DNA-damaging agents. Testicular cells were chosen because the germinal epithelium of testes is one of the most proliferately active tissues potentially susceptible to DNA-damaging effects. As a second target peripheral blood lymphocytes were chosen because dietary lignin or its metabolites circulate in the animal organism probably through the blood system. For the in vitro experiments, isolated cells were preincubated with lignin for 2 h before treatment with one of the oxidative agents. In ex vivo experiments, the cells were exposed to H2O2 or visible light-excited MB after isolation from rats fed either a common diet or a lignin-supplemented diet. The water-soluble, sulfur-free lignin used in experiments was obtained by fractionation of hardwood hydrolysate. The level of direct single-strand DNA breaks in H2O2-treated cells was measured by the classical comet assay, and the level of oxidative DNA lesions in visible light-treated cells was measured by a modified comet assay. We found that lignin reduced DNA lesions induced by H2O2 or visible light-excited MB both in vitro and ex vivo. The major conclusion of our study is that lignin polymer obtained by fractionation of hardwood hydrolysate manifested a specific type of antimutagenic effect.