Mitochondrial (mt) DNA from four sibling species within the Paramecium aurelia complex, including stocks of different geographic origin and mutants, were analyzed using four 6-bp recognition site and one 4-bp recognition site endonucleases and the sequence divergence was estimated using Upholt's (1977) statistical procedure. All four species were readily distinguishable regardless of the restriction endonuclease employed. With intraspecies comparisons, no differences were observed which could be accounted for on the basis of geographic origin. Except for species 4, each stock (and mutant) gave a species-specific fragment pattern. For species 4, while the patterns were distinct from the other species, two species-specific type of patterns were found, designated A and B. The sequence divergence between these was estimated to be between 1 and 2 percent. With interspecies comparisons, the sequence divergence ranged from 3.9 to 10.3% with the greatest divergence being between species 1 and 4, and the least between species 1 and 5. The similarity between species 1 and 5 is in accord with other criteria for interspecies comparisons. The degree of sequence divergence measured here in Paramecium mt DNA is well within the range reported for rodents and primates. All four species mt DNA were cleaved to many DNA fragments by DPN II, an enzyme which recognizes non-methylated sites, and not by DPNI, the methyl-site specific counterpart of DPN II, suggesting that mt DNA from Paramecium aurelia is not appreciably methylated, if at all.