Pseudomonas pseudoflava GA3, an aerobic hydrogen-oxidizing bacterium, was characterized physiologically. The doubling time of autotrophically growing cells was 2.5 h for cells growing heterotrophically on sucrose it was 1.5 h; and for cells growing heterotrophically on fructose, succinate, lactate, glutamate, treahalose, glucose, and pyruvate it was about 1.6 h. Glucose and fructose were degraded via the Entner-Doudoroff pathway. 6-Phosphogluconate dehydrogenases (NAD or NADP-dependent) were not detected. Hydrogen did not suppress or inhibit the utilization of organic substrates. The specific hydrogenase activity was influenced by the growth conditions. An oxygen concentration of 20% (vol/vol) in the gas atmosphere provided conditions for continued exponential growth but diminished the specific hydrogenase activity. Cells contained a single membrane-bound hydrogenase; intact membranes were able to reduce menadion, ubiquinone Q10, methylene blue, phenazine methosulfate, and dichlorophenolindophenol. The hydrogenase was solubilized from the membrane fraction by treatment with 0.2% Triton X-100 and 10% sucrose, resulting in a yield of about 60% of total activity.