It has been widely accepted that most coenzyme Q (CoQ) exists freely in the mitochondrial membrane as a CoQ pool. However, the recent identification of a mitochondrial CoQ-binding protein, termed Coq10, in budding yeast has the potential to change our current view of CoQ status in membranes. Here, we studied the counterpart of budding yeast Coq10 (also termed Coq10) in fission yeast. Fission yeast coq10 null mutants exhibited a similar, but less severe, phenotype to CoQ-deficient fission yeast, including the requirement for antioxidants for proper growth on minimal medium, increased sensitivity to H(2)O(2), high levels of H(2)S production, and a deficiency in respiration. The coq10 null mutant produced nearly normal levels of CoQ10, suggesting that coq10 does not belong to the group of CoQ biosynthetic genes. To elucidate the role of Coq10, we expressed recombinant coq10 in Escherichia coli, and found that CoQ8 was present in purified recombinant Coq10. Mutational analysis of 13 conserved residues of Coq10 revealed that two hydrophobic amino acid residues, leucine 63 (L63) and tryptophan 104 (W104), play an important role in Coq10 binding to CoQ. An L63A/W104A double mutant of Coq10 exhibited lower CoQ-binding activity than either of the single mutants, and was unable to complement the coq10 deletion in fission yeast. A human Coq10 ortholog was able to functionally compensate for the absence of coq10 in fission yeast, suggesting that Coq10 is important for proper respiration in a variety of organisms.