The 3243A>G mutation in the MTTL1 (tRNA(Leu)) gene and the 8344A>G mutation in the MTTK (tRNA(Lys)) gene are the most common mutations found in mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes and myoclonic epilepsy associated with ragged-red fibers, respectively. These mitochondrial DNA mutations are usually detected by conventional polymerase chain reaction followed by restriction enzyme digestion and gel electrophoresis. We developed a LightCycler real-time polymerase chain reaction assay to detect these two mutations based on fluorescence resonance energy transfer technology and melting curve analysis. Primers and fluorescence-labeled hybridization probes were designed so that the sensor probe spans the mutation site. The observed melting temperatures differed in the mutant and wild-type DNA by 9 degrees C for the MTTL1 gene and 6 degrees C for the MTTK gene. This method correctly identified all 10 samples that were 3243A>G mutation-positive, all 4 samples that were 8344A>G mutation-positive, and all 30 samples that were negative for both mutations, as previously identified by traditional gel-based methods. This LightCycler assay is a rapid and reliable technique for molecular diagnosis of these mitochondrial gene mutations.