The biosynthesis of ubiquinone (coenzyme Q) was studied in Saccharomyces cerevisiae. Lipid extracts were prepared from wild-type yeast grown in the presence of p-[U-14C]- and p-[carboxy-14C]hydroxybenzoic acid. Ergosterol was removed by adsorption to digitonin-celite, and radiolabeled lipids were purified by sequential reverse-phase and normal-phase HPLC steps. Radiolabeled peaks were identified by comparison with synthetic standards using retention time and electron ionization mass spectrometric criteria. The recovery and identification of the unstable 3-hexaprenyl-4-hydroxybenzoic acid molecule were facilitated by treatment of the lipid extract with diazomethane under conditions that resulted in the formation of the stable derivatives methyl 3-hexaprenyl-4-hydroxybenzoate or methyl 3-hexaprenyl-4-methoxybenzoate. In stationary-phase yeast cultures, the major radioactive lipid products are coenzyme Q and 3-hexaprenyl-4-hydroxybenzoic acid, constituting 62 and 38% of the radioactive lipids, respectively. However, under log-phase growth conditions the biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid predominates (accounting for 81% of the radioactive lipids). The data indicate that in wild-type yeast, 3-hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis and that in log-phase growth this ubiquinone intermediate is present at fourfold higher abundance than the end product. The physiological rationale for this high concentration of a membrane-bound intermediate is unclear.