To date, direct analysis of mitochondrial proteomes has largely been limited to animals, fungi and plants. To broaden our knowledge of mitochondrial structure and function, and to provide additional insight into the evolution of this key eukaryotic organelle, we have undertaken the first comprehensive analysis of the mitochondrial proteome of a protist. Highly purified mitochondria from Tetrahymena thermophila, a ciliated protozoon, were digested exhaustively with trypsin and the resulting peptides subjected to tandem liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS). In this way, we directly identified a total of 573 mitochondrial proteins, 545 of which are encoded by the nuclear genome and 28 by the mitochondrial genome. The latter number includes a novel, 44 residue protein (which we designate Ymf78) that had not been recognized during annotation of the T. thermophila mtDNA sequence. The corresponding gene, ymf78, is highly conserved in genomic position, size and sequence within the genus Tetrahymena. Our analysis has provided broad coverage of both membrane-bound and soluble proteins from the various submitochondrial compartments, with prominent representatives including components of the tricarboxylic acid cycle, Complexes I-IV of the electron transport chain and Complex V (ATP synthase), the mitochondrial transcription and translation machinery, the TOM and TIM protein translocases, various mitochondrial transporters, chaperonins (Cpn60, Hsp70, Hsp90), at least four FtsH family ATP-dependent metalloproteases implicated in m-AAA and i-AAA protease function, and enzymes involved in lipid, amino acid and coenzyme metabolism, as well as iron-sulfur cluster formation. Unexpectedly, six of the ten enzymes of glycolysis were found by MS analysis of purified T. thermophila mitochondria, whereas no hits were seen to any cytosolic ribosomal proteins. At least one of the glycolytic proteins, enolase, has an evident N-terminal extension that exhibits characteristics of a typical mitochondrial targeting peptide. As in other organisms, phylogenetic analysis of functionally annotated mitochondrial proteins demonstrates that <20% can be traced confidently to the alpha-proteobacterial lineage of Bacteria, emphasizing the chimeric evolutionary nature of the mitochondrial proteome. Notably, about 45% of the proteins identified in our analysis have no known function, and most of these do not have obvious homologs outside of the ciliate lineage. About two-thirds of these ORFan proteins have putative homologs in another ciliate, Paramecium tetraurelia, whereas the remainder appear to be Tetrahymena-specific. These results emphasize the power and importance of direct MS-based analysis of mitochondria in revealing novel mitochondrial proteins in different eukaryotic lineages. Our observations reinforce an emerging view of the mitochondrion as an evolutionarily flexible organelle, with novel proteins (and presumably functions) being added in a lineage-specific fashion to an ancient, highly conserved functional core, much of which was contributed by the presumptive alpha-proteobacterial symbiont from which the mitochondrial genome was derived.