Phenyl-substituted dihydropyrazines (Ph-DHPs) are derivatives of 2,3-dihydro-5,6-dimethylpyrazine (Me-DHP). Upon the addition of Cu(2+), Me-DHP inhibits the growth of Escherichia coli by generating hydroxyl and carbon-centered radicals that cause DNA strand breakage. Here, we investigated the toxic effect of Ph-DHPs in several DNA repair-deficient or detoxifying enzyme-deficient mutant strains. Ph-DHPs caused cytotoxic and genotoxic damage, but, in a sodA sodB strain, the effects in the presence or absence of Cu(2+) were different than those of Me-DHP. Our results suggest that the action of the generated superoxide anion in the interior side of the cell is remarkable.