The oxidation of glucose by Pseudomonas aeruginosa is known to follow the sequence: glucose→ gluconic acid→2-ketogluconic acid→pyruvic acid and thence into the tricarboxylic acid cycle. The most striking aspect of this pathway is that the first two oxidative steps do not involve phosphorylated intermediates at the substrate level. In the present study radioactive phosphorus was used in an attempt to elucidate the carbohydrate metabolism of P. aeruginosa. Cell free preparations of P. aeruginosa, obtained by crushing a cell paste in the Hughes press, incubated with added cofactors, ADP and P³² resulted in the formation of labelled ADP and ATP. The presence of glucose or succinate in the reaction mixture greatly depressed the amount of ATP found. The cell free preparations were found to yield ATP as measured in the hexokinase trap, but the formation of ATP was not increased by the addition of glucose, gluconic acid, 2-ketogluconic acid or succinic acid. These results suggested that no net energy was gained by the extract by the oxidation glucose→ gluconic acid→ 2-ketogluconic acid. In manometric experiments it was found that the cell free preparation did not oxidize glucose-6-phosphate, ribose-5-phosphate, α-ketoglutarate, citrate and isocitrate. Glucose was oxidized with the uptake of two atoms of oxygen per mole of substrate. In the presence of ATP, glucose was oxidized with the uptake of only one atom of oxygen. Gluconic acid and gluconolactone were oxidized with the uptake of one atom of oxygen; ATP had no effect on these last two oxidations. From these data two reactions beyond 2-ketogluconate have been postulated. [Formulas omitted] / Science, Faculty of / Microbiology and Immunology, Department of / Graduate