The location and function of ubiquinone in the electron transport system of Pseudomonas aeruginosa grown aerobically were studied. The reduction level of ubiquinone in the intact membrane was 36-43% in the aerobic steady state and about 65% in the anaerobic state with one substrate, but the level in the anaerobic state reached to 81% with a mixture of several substrates. Complete removal of ubiquinone performed by extracting the lyophilized membrane particles with n-pentane containing acetone resulted in complete loss of all oxidase activities for glucose, gluconate, malate, succinate, and NADH. In the ubiquinone-depleted particles, neither cytochrome component was reduced by adding any substrate. Reincorporation of coenzyme Q9 into the depleted particles restored each oxidase activity to 60 to 80% of the original and reduction of cytochromes with substrates. The reduction kinetics of cytochromes and effect of inhibitors showed that coenzyme Q9 was incorporated at the original site in the electron transport system. Exogenous coenzyme Q2 increased gluconate and malate oxidase activities and decreased glucose oxidase activity, when French-pressed membrane vesicles but not spheroplasts were used. Oxidizing activity for reduced coenzyme Q2 was also detected in the pressed vesicles but not in the spheroplasts. The present results showed that ubiquinone was indispensable and located prior to cytochromes in the electron transport system. Furthermore, the homogeneity and sidedness of ubiquinone in the cytoplasmic membrane of the organism are also discussed.