We report the effect on complex I function of the 14484 Leber's hereditary optic neuropathy (LHON) mutation affecting the ND6 subunit gene. The same gene was also reported to carry another mutation, at position 14459, associated with the LHON/dystonia phenotype that induces a reduction of complex I-specific activity and increases the sensitivity to the product decylubiquinol. Given the proximity of both mutations in the ND6 gene, we tested the specific activity of complex I and its sensitivity to myxothiazol and nonylbenzoquinol, both inhibitors at the ubiquinol product site, in platelet submitochondrial particles from nine 14484 homoplasmic individuals, 8 Italians with Caucasian mtDNA haplogroup J (adjunctive 4216 and 13708 mutations), and 1 Tunisian with an African mtDNA haplogroup. The specific activity of complex I was not affected by the 14484 mutation, but the sensitivity to both inhibitors was significantly increased compared with control subjects regardless of the presence of haplogroup J polymorphisms. Analysis of 70 different amino acid sequences of the ND6 subunit indicated that the 14484 mutation affects an amino acid belonging to its most conserved region, which shows local similarities with cytochrome b regions interacting with ubiquinone or ubiquinol in complex III. Our results suggest that both 14484 and 14459 mutations may affect amino acids forming the interaction site of ubiquinol product, and the 14484 mutation produces a biochemical defect resembling in part that already reported for the common 11778/ND4 LHON mutation.