We report here the development of transgenic mice with mtDNA with a deleterious mutation (‘transmitochondrial mice’). To study segregation and transmission of mtDNA mutants, we have generated a female embryonic stem (ES) cell line that is heteroplasmic for normal (chloramphenicol-sensitive or wild-type mtDNA) and chloramphenicol-resistant (CAPr) mtDNA. As donor CAPr mtDNA was originally derived from mouse 3T3 cells that are chloramphenicol-resistant because of an AT substitution at bp 2,379 in the mitochondrial 16S rRNA gene10, any wild-type mtDNA in CAPr ES cells could potentially be of two distinctive lineages. Chimeric mice should have three populations of mtDNA: wild-type and CAPr mtDNA in the ES cells and wild-type mtDNA in the cells derived from the recipient blastocyst. This contrasts with the founder mice in the previous models of mtDNA heteroplasmy, in which every cell is populated from the same two mtDNA populations. The A2379T mutation increases lactate production in LA9 cells11, indicative of a respiratory chain defect. CAPr 3T3 cells were enucleated and fused to ES cells. The proportion of CAPr mtDNA in the resulting ES lines was greater than 90% and the cells manifested reduced cytochrome oxidase activity on cytochemical staining12 compared with that of wild-type ES cells (Fig. 1). These ES cells were injected into blastocysts using standard microinjection technology.