At present, there are no means for creation of relevant animal models of human mitochondrial DNA (mtDNA)-based diseases in a directed fashion. As an initial step towards this end, we have developed a microinjection technique for transfer of isolated, viable mitochondria between two mouse species. Previously, we reported detection, by nested PCR with species-specific primer sets, of Mus spretus mtDNA in Mus musculus domesticus blastocyts following zygote microinjection and culture. We now report the production of transmitochondrial founder mice, and germline transmission of the heteroplasmic state in a maternal lineage. Heteroplasmic mice produced by this technique will be useful in the study of mitochondrial dynamics and may hasten the creation of animal models of human mtDNA-based diseases.