Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis.