In this study, the modulating effect of L-carnitine on tert-butyl-hydroperoxide-induced DNA damage was compared with that of mannitol, a well known scavenger of hydroxyl radicals, both in normal and Ataxia telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines established from A. telangiectasia (A-T) patients. The alkaline version of the comet assay was employed to measure the frequency of single-strand breaks (SSBs) and alkali-labile sites induced by t-butyl-OOH immediately after treatment and at different recovery times in normal and A-T cell lines, with and without pre-treatment with L-carnitine. In addition, both the yield of induced chromosomal damage and the effect on cell proliferation were evaluated. Our results show that pre-treatment of cells with L-carnitine produced an enhancement of the rate and extent of DNA repair in A-T cell lines at early recovery time; furthermore, in samples pre-treated with L-carnitine a reduction of all types of chromosomal aberration was observed, both in A-T and in wild-type cell lines. The reducing effect of L-carnitine pre-treatment on oxidative DNA damage was more prominent than that of pre-treatment with mannitol. In conclusion, we demonstrated a protective effect of L-carnitine on oxidative stress-induced DNA damage in A-T cells, suggesting its possible role in future pharmacological applications in A-T therapy.