The ubiquinone precursors, 2-octaprenyl-6-methoxy-1,4-benzoquinone and 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone, were isolated from ubiquinone-deficient mutants of Escherichia coliand identified by nuclear magnetic resonance and mass spectrometry. Mutants accumulating 2-octaprenyl-6-methoxy-1,4-benzoquinone and 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone were shown to carry mutations in genes designated ubiEand ubiF, respectively. The ubiEgene was shown to be cotransducible with metE(minute 75) and close to two other genes concerned with ubiquinone biosynthesis. The ubiFgene was located close to minute 16 by cotransduction with the lip, gltA, and entAgenes.