The glucose metabolism of Fibrobacter succinogenes S85 was studied in detail; key intermediates and alternative pathways were evidenced by NMR and/or enzymatic assays. A high phosphoketolase activity was detected in four strains of Fibrobacter under strictly anaerobic conditions, with ribose-5-phosphate as substrate, no activity was evidenced with fructose-6-phosphate. This is the first report of a pentose phosphates phosphoketolase in bacteria unable to use pentoses. In contrast, the Entner-Doudoroff pathway and the oxidative branch of the pentose phosphate pathway could not be evidenced. Incubation of living cells of F. succinogenes with Na2(13)CO3 confirmed the incorporation of 13CO2 in the carboxylic group of succinate. The presence of fumarase was evidenced by in vivo 4C-NMR using 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO): the enzyme showed a high reversibility under physiological conditions. The production of formate from glucose catabolism was evidenced by enzymatic assay and by NMR and a pyruvate formate lyase activity was detected using strictly anaerobic conditions.