Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of thep-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10synthesis, we investigated CoQ10production in 400Schizosaccharomyces pombegene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion ofcoq11(anS. cerevisiaeCOQ11homolog) and a novel gene designatedcoq12lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB orp-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of theΔcoq12strain, but these compounds did not affect theΔcoq11strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+reductase domain. We determined that purified Coq12 protein fromS. pombedisplayed NAD+reductase activity when incubated with ethanol-extracted substrate ofS. pombe. Because purified Coq12 fromEscherichia colidid not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC–MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.