Eighteen Italian patients presenting with sporadic, bilateral, simultaneous, or sequential optic neuritis (ON) were evaluated for 14 base changes in mitochondrial DNA (mtDNA) previously found associated with Leber's hereditary optic neuropathy (LHON), aiming to identify at a molecular level LHON cases with nontypical phenotypes. During a 36-month follow-up, 11 ON patients developed clinical or laboratory features allowing diagnosis of clinically definite multiple sclerosis (MS). None was positive for any of the "primary" LHON-associated mutations. However, single or multiple "secondary" LHON-associated sequence changes at 4216/ND1, 4917/ND2, and 13708/ND5 were detected in ON and ON-MS patients. MS controls without visual failure as well as healthy control subjects harbored the same base changes at similar frequencies. In addition, coexistence of three sequence changes was found in two cases (1 ON-MS patient and 1 MS control patient). We also report finding two new neutral sequence base changes in the ND-4 gene which were identified by SSCP and confirmed by automated DNA sequence analysis. The results suggests that these secondary mutations do not contribute to MS susceptibility in these patients, but rather represent neutral mitochondrial DNA polymorphisms. In addition, whether there are biochemical abnormalities related to single and multiple secondary mtDNA sequence changes remain to be demonstrated.