Defects of the mitochondrial respiratory chain in cardiac muscle are an important, yet still overlooked cause of heart failure. In 16 of 32 endocardial biopsies from infants affected by “idiopathic” hypertrophic cardiomyopathy we demonstrated a remarkable decrease of activity of either complex I, or complex IV, or both, relative to complex II + III activity which was taken as an index of mitochondrial proliferation. At the molecular level, several mtDNA mutations have been associated with cardiomyopathy. For instance, MIMyCa is a maternally inherited syndrome presenting with a variable combination of skeletal and heart muscle failure associated with a heteroplasmic A3260G transition in the tRNALeu(UUR) gene. To study the effects of the mutation in a controlled system, we prepared clones of transmitochondrial cybrids by fusing mutant cytoplasts with mtDNA-less tumor cells. Two groups of clones were identified: nearly 100% mutant (M group) and nearly 100% wildtype (WT group). The means of complex I and IV in the M group were 63% and 67% relative to the WT group. The O2 consumption in the M group was 36%, and the lactate production was 218% of that in the WT group. MtDNA-specific translation was defective in M clones. The study of transmitochondrial cybrids is an important clue to test the pathogenicity of mtDNA mutations. © 1995 John Wiley & Sons, Inc.