Cell-free preparations of Acetobacter melanogenum readily oxidized glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, 6-phosphogluconate, and ribose-5-phosphate; fructose-1,6-diphosphate was utilized very slowly. The presence of an active triphosphopyridine nucleotide (TPN)-linked glucose-6-phosphate dehydrogenase and of phosphohexose isomerase was demonstrated; phosphoglucomutase was also present in these extracts. 6-Phosphogluconate dehydrogenase activity (TPN-linked) was low in extracts of glucose-grown cells, but was high in sonates of gluconate-grown cells. An active oxidizing system for reduced diphosphopyridine nucleotide was present but transhydrogenase was not detected. Pyruvate was produced extensively from 6-phosphogluconate but only slowly from ribose-5-phosphate; fluoride inhibited pyruvate formation from both substances. Ribose-5-phosphate was degraded readily via the transketolase-transaldolase series of reactions. Glyceraldehyde-3-phosphate dehydrogenase, hexokinase, gluconokinase, 2-ketogluconokinase, and aldolase were detected but not phosphohexokinase. The results suggest that glucose is oxidized either directly or after phosphorylation but is not metabolized glycolytically. The oxidation of hexose phosphate appears to occur predominantly as a result of the splitting of 6-phosphogluconate to pyruvate and glyceraldehyde-3-phosphate.