In the present work, a large scale investigation was done regarding the capacity of cultured human cell lines (carrying in homoplasmic form either the mitochondrial tRNA(Lys) A8344G mutation associated with the myoclonic epilepsy and ragged red fiber (MERRF) encephalomyopathy or a frameshift mutation, isolated in vitro, in the gene for the ND4 subunit of NADH dehydrogenase) to undergo transcomplementation of their recessive mitochondrial DNA (mtDNA) mutations after cell fusion. The presence of appropriate nuclear drug resistance markers in the two cell lines allowed measurements of the frequency of cell fusion in glucose-containing medium, non-selective for respiratory capacity, whereas the frequency of transcomplementation of the two mtDNA mutations was determined by growing the same cell fusion mixture in galactose-containing medium, selective for respiratory competence. Transcomplementation of the two mutations was revealed by the re-establishment of normal mitochondrial protein synthesis and respiratory activity and by the relative rates synthesis of two isoforms of the ND3 subunit of NADH dehydrogenase. The results of several experiments showed a cell fusion frequency between 1.4 and 3.4% and an absolute transcomplementation frequency that varied between 1.2 x 10(-5) and 5.5 x 10(-4). Thus, only 0.3-1.6% of the fusion products exhibited transcomplementation of the two mutations. These rare transcomplementing clones were very sluggish in developing, grew very slowly thereafter, and showed a substantial rate of cell death (22-28%). The present results strongly support the conclusion that the capacity of mitochondria to fuse and mix their contents is not a general intrinsic property of these organelles in mammalian cells, although it may become activated in some developmental or physiological situations.