Abstract The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1–1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH-depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM y-BuOOH were repaired within 60 min of treatment in both GSH-depleted (GSH −) and non-depleted (GSH +) cells. In contrast, chromosomal aberrations increased a little during the 60 min after treatment in both GSH − and GSH + cells. The aberrations were then repaired in GSH + cells but those in GSH − cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.