Ubiquinone (coenzyme Q; abbreviation, Q) plays an essential role in electron transport in Escherichia coli when oxygen or nitrate is the electron acceptor. The biosynthesis of Q involves at least nine reactions. Three of these reactions involve hydroxylations resulting in the introduction of hydroxyl groups at positions C-6, C-4, and C-5 of the benzene nucleus of Q. The genes encoding the enzymes responsible for these hydroxylations, ubiB, ubiH, and ubiF are located at 87, 66, and 15 min of the E. coli linkage map. The ubiF encoded oxygenase introduces the hydroxyl group at carbon five of 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol resulting in the formation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1, 4-benzoquinol. An ubiF mutant failed to carry out this conversion. Based on the homology to UbiH, an open reading frame (orf391) was identified at the 15 min region of the chromosome, amplified using PCR, and cloned into pUC18 plasmid. The ubiF mutants, when complemented with this plasmid, regained the ability to grow on succinate and synthesize Q.