The effect of the presence of ammonia on [1-¹³C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by ¹³C and ¹H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The ¹³C enrichment of succinate and acetate was precisely quantified by ¹³C-filtered spin-echo difference ¹H-NMR spectroscopy. The presence of ammonia did not modify the ¹³C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the ¹³C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by ¹³C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and ¹H NMR. When cells were incubated in vivo with [1-¹³C]glucose, only ¹³C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.