Abstract: The ubiquinone coenzyme Q (CoQ) is synthesized in mitochondria with a large, hydrophobic isoprenoid side chain. It functions in mitochondrial respiration as well as protecting membranes from oxidative damage. Yeast that cannot synthesize CoQ (DeltaCoQ) are viable, but cannot grow on nonfermentable carbon sources, unless supplied with ubiquinone. Previously we demonstrated that the isoprenoid side chain of the exogenous ubiquinone was important for growth of a DeltaCoQ strain on the nonfermentable substrate glycerol [James AM et al. (2005) J Biol Chem280, 21295-21312]. In the present study we investigated the structural requirements of exogenously supplied CoQ(2) for growth on glycerol and found that the first double bond of the initial isoprenoid unit is essential for utilization of respiratory substrates. As CoQ(2) analogues that did not complement growth on glycerol supported respiration in isolated mitochondria, discrimination does not occur via the respiratory chain complexes. The endogenous form of CoQ in yeast (CoQ(6)) is extremely hydrophobic and transported to mitochondria via the endocytic pathway when supplied exogenously. We found that CoQ(2) does not require this pathway when supplied exogenously and the pathway is unlikely to be responsible for the structural discrimination observed. Interestingly, decylQ, an analogue unable to support growth on glycerol, is not toxic, but antagonizes growth of DeltaCoQ yeast in the presence of exogenous CoQ(2). Using a DeltaCoQ double-knockout library we identified a number of genes that decrease the ability of yeast to grow on exogenous CoQ. Here we suggest that CoQ or its redox state may be a signal for growth during the shift to respiration.