To quantify the amount of the 3460G-->A/ND1 point mutation responsible for Leber's hereditary optic neuropathy, we developed a quantitative real-time polymerase chain reaction method based on the SYBR Green assay and a new approach using the TaqMan assay. Both methods were based on the amplification refractory mutation system, comparing the heteroplasmic load quantified by restriction fragment length polymorphism in 15 Leber's hereditary optic neuropathy family members, with the results obtained using quantitative real-time polymerase chain reaction methods. The comparative evaluation of mitochondrial DNA (mtDNA) heteroplasmy from blood samples showed significant correlation between restriction fragment length polymorphism analysis, real-time SYBR Green assay, and TaqMan assay. We validated the last method by measuring experimental samples composed by a known proportion of cloned plasmids containing either the wild-type or mutant sequence, giving a correlation coefficient of 0.999 (P < 0.0001). The real-time amplification refractory mutation system polymerase chain reaction by TaqMan assay provides a rapid, reliable, sensitive, reproducible, and one-step quantitative method to detect heteroplasmic mutant mtDNA. This method allows the quantitation of a broad range of mutational load (up to 100%, down to 0.01%) on the basis of in vitro calibration, thus rendering the TaqMan assay suitable for the diagnostic analysis of heteroplasmic load in mtDNA-related disorders.