Ubiquinone, coenzyme Q, plays a pivotal role in electron transport and is a target for chemotherapy against a number of eukaryotic infectious agents, including Pneumocystis carinii. Coenzyme Q₁₀ was previously identified as the major ubiquinone homolog in P. carinii isolated and purified from rat lungs; CoQ₉ was also present. In contrast, CoQ₉ and CoQ₈ (but not CoQ₁₀) were detected in the lungs of uninfected rat controls. These observations suggested that the pathogen synthesizes CoQ₁₀, and perhaps CoQ₉ as well. In the present study, CoQ biosynthesis in P. carinii was examined in greater detail. Radiolabeled mevalonate, a precursor of the CoQ polyprenyl chain, was incorporated in vitro into P. carinii ubiquinones. Incorporation of radiolabeled mevalonate into P. carinii CoQ was not enhanced by treating cells with lovastatin, suggesting that the cells did not transport the drug, or that a lovastatin- insensitive pathway for de novo synthesis of isoprenoids may also function in this organism. Radiolabeled precursors of the ring moiety, including shikimic acid, p-hydroxybenzoic acid, and tyrosine were also incorporated into P. carinii CoQ. Unexpectedly, it was found that not only CoQ₉ and CoQ₁₀, but also CoQ₇, and CoQ₈, were metabolically radiolabeled by all the precursors tested, indicating that the organism synthesizes CoQ₇, CoQ₈, CoQ₉, and CoQ₁₀. Metabolic radiolabeling of ubiquinones in rat lung controls was not detected in experiments using either radioactive mevalonate or p-hydroxybenzoate. Thus the incorporations measured using purified P. carinii preparations were due to the enzymes of the organism.