We report a method for introducing mtDNA mutations into the mouse female germ line by means of embryonic stem (ES) cell cybrids. Mitochondria were recovered from the brain of a NZB mouse by fusion of synaptosomes to a mtDNA-deficient (ρ°) cell line. These cybrids were enucleated and the cytoplasts were electrofused to rhodamine-6G (R-6G)-treated female ES cells. The resulting ES cell cybrids permitted transmission of the NZB mtDNAs through the mouse maternal lineage for three generations. Similarly, mtDNAs from a partially respiratory-deficient chloramphenicol-resistant (CAP^R) cell line also were introduced into female chimeric mice and were transmitted to the progeny. CAP^R chimeric mice developed a variety of ocular abnormalities, including congenital cataracts, decreased retinal function, and hamaratomas of the optic nerve. The germ-line transmission of the CAP^R mutation resulted in animals with growth retardation, myopathy, dilated cardiomyopathy, and perinatal or in utero lethality. Skeletal and heart muscle mitochondria of the CAP^R mice were enlarged and atypical with inclusions. This mouse ES cell-cybrid approach now provides the means to generate a wide variety of mouse models of mitochondrial disease.