In a previous study we screened a range of mushroom species growing in Slovenia for their anti-genotoxic potential and found Lactarius vellereus to be the most effective. In this study genotoxic and anti-genotoxic activities of methanol extracts of Lactarius vellereus (Fr.: Fr.) Fr. were evaluated in the bacterial reverse mutation test with Salmonella typhimurium TA98 and, in the mammalian cell test with human hepatoma (HepG2) cells, using the comet assay to measure DNA damage. The extract induced no mutations in S. typhimurium TA98 and no DNA damage in HepG2 cells. Against the indirect acting mutagen 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) the extract showed significant, dose dependent antimutagenic activity, while it did not counteract the direct acting mutagen 4-nitroquinoline oxide (4-NQO). The extract also exerted a protective effect against IQ induced genotoxicity in mammalian cells of human origin. Treatment of HepG2 cells with the L. vellereus extract (125-500 microg/ml) together with IQ, reduced the genotoxic effect of the latter in a dose dependent manner. Our findings show that a methanol extract of L. vellereus is highly protective against IQ induced DNA damage in human derived cells and L. vellereus can be considered as a natural source of antimutagens with potential pharmacological applications in cancer prevention.