Increasing concentrations of glucose (1-5%) in the growth medium depressed ubiquinone-6 biosynthesis in continuously cultured wild type Saccharomyces cerevisiae. In addition, an early intermediate in the pathway of ubiquinone-6 biosynthesis, i.e. 3,4-dihydroxy-5-hexaprenylbenzoate (3,4-DHHB), was found to accumulate. The increase in 3,4-DHHB levels varied inversely with the diminished levels of ubiquinone-6, suggesting that O-methylation of 3,4-DHHB is a regulated step in catabolite repression. Experiments using protoplasts demonstrated that the effect of catabolite repression on this pathway was reversible by 1.2 mM cAMP but not by other nucleotides and cyclic nucleotides. This response to cAMP was unaltered by the protein synthesis inhibitor cycloheximide, indicating that the regulatory control for this reaction must occur at the enzymatic level. Additional experiments demonstrated the presence of a heat-labile component of the cytoplasm, which was essential for this effect of cAMP. This observation suggests that this cytosolic effector may be translocated to the inner membrane of the mitochondria, the intracellular site for ubiquinone-6 biosynthesis.