The mitochondrial cytochrome c oxidase 1 (CO1) genes of two isolates of each of the seven mating types of Tetrahymena thermophila were sequenced and found to differ by < 1% in nucleotide sequence and to be identical by putative protein sequence. As this gene was highly conserved in this species, the CO1 gene sequence was determined for four pairs of Tetrahymena species identical in their small subunit rRNA gene sequences. The following pairs of species showed from 1% to 12% divergence at the nucleotide level, enabling discrimination of all these species: (1) Tetrahymena pyriformis strain T and Tetrahymena setosa strain HZ-1; (2) Tetrahymena canadensis strain UM1215 and Tetrahymena rostrata strain ID-3; (3) Tetrahymena pigmentosa strain UM1285 and Tetrahymena hyperangularis strain EN112; and (4) Tetrahymena tropicalis strain TC-105 and Tetrahymena mobilis. However, because of the synonymous nature of the majority of substitutions, the pairs of species were identical based on the putative protein sequence.