Primary mitochondrial DNA (mtDNA) mutation at the nt 11778 site in Leber's hereditary optic neuropathy (LHON) has been reported to be present in patients with ethambutol-induced optic neuropathy. To study further this association between LHON and ethambutol-induced optic neuropathy, we tested ethambutol-induced optic neuropathy patients for the presence of the mtDNA mutations at nucleotides (nt)–11778, nt–14484, nt–3460, nt–15257, nt–9438, nt–9804, nt–13730, and nt–14459 in 24, 15, 8, 6, 5, 5, 5, and 5 patients respectively. However, none of the ethambutol-induced optic neuropathy patients was found to exhibit any pathogenic LHON mtDNA mutation. In conclusion, we found no evidence of any association between ethambutol-induced optic neuropathy and the LHON mutations.