The large majority of mitochondrial DNA (mtDNA) deletions analyzed from mitochondrial myopathies and aging humans have been found to be flanked by direct repeats, a finding which has led to the slip-replication hypothesis of deletion formation. In this study, we have characterized 13 mtDNA deletion breakpoints from skeletal muscle harvested from 9- to 27-year-old rhesus monkeys. Seven of the deletions, five of which were unique to a particular animal, did not have direct repeats at the deletion breakpoints. In contrast, two of the three deletions common to several animals had direct repeats flanking the breakpoints. It appears, therefore, that at least two different mechanisms exist by which mtDNA deletions are formed during aging, one requiring and one independent of flanking direct repeats. Furthermore, the species in which mtDNA deletions are detected may determine which mechanism predominates.