The recent development of cellular models of mitochondrial DNA-linked diseases by transfer of patient-derived mitochondria into human mtDNA-less (rho o) cells has provided a valuable tool for investigating the complementation and segregation of mtDNA mutations. In transformants carrying in heteroplasmic form the mitochondrial tRNA(Lys) gene 8344 mutation or tRNA(Leu(UUR)) gene 3243 mutation associated, respectively, with the MERRF or the MELAS encephalomyopathy, full protection of the cells against the protein synthesis and respiration defects caused by the mutations was observed when the wild-type mtDNA exceeded 10% of the total complement. In the MERRF transformants, the protective effect of wild-type mtDNA was shown to involve interactions of the mutant and wild-type gene products, probably coexisting within the same organelle from the time of the mutation event. In striking contrast, in experiments in which two mtDNAs carrying either the MERRF or the MELAS mutation were sequentially introduced within distinct organelles into the same rho o cells, no evidence of cooperation between their products was observed. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which a chloramphenicol (CAP) resistance-conferring mtDNA mutation was introduced into CAP-sensitive cells. In the area of segregation of mtDNA mutations, in unstable heteroplasmic MELAS transformants, observations were made which pointed to a replicative advantage of mutant molecules, leading to a rapid shift of the genome towards the mutant type. These results are consistent with a model in which the mitochondrion, rather than the mtDNA molecule, is the segregating unit.