Mitochondrial DNA (mtDNA) is a multicopy, maternally inherited, genome. Individuals frequently carry a mixture of genetically distinct mtDNA molecules whose proportions may vary between sexual generations or among tissues from the same individual. Analyses of the genetic composition of mitochondria have previously relied on electron microscopy and have not permitted the genotype of single mitochondria to be determined. We have developed flow cytometry techniques to isolate single mitochondrial particles and PCR-based assays to determine the mtDNA copy number and composition of individual particles. In a first application of this method, we studied mitochondrial particles from fibroblast cells heteroplasmic for the tRNA lys(8344) point mutation, associated with myoclonus epilepsy and ragged red fiber (MERRF). Individual mitochondrial particles contained between 0 and 11 mtDNA molecules with a mean of 2.0 (95% CI 1.6-2.4). The majority (75%) of the mitochondrial particles from which a PCR product was obtained contained only one type of mtDNA, consistent with the low mean mtDNA copy number. The method developed may be applied to studies of the copy number and distribution of mtDNA genomes in different cell types.