Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6- phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PP(i). Phosphoglycerate kinase, pyruvate kinase, and acetate kinase coupled GDP as well as ADP, but P(i) compounds were not their phosphoryl acceptor. Cell extracts showed GDP- dependent activity of phosphoenolpyruvate carboxykinase, which assimilates bicarbonate and phosphoenolpyruvate into oxaloacetate, a precursor of succinate. Considerable amounts of GTP, polyphosphate, and PP(i) were found in glucose-fermenting cells, indicating that these compounds may serve as phosphoryl donors or acceptors in Actinomyces cells. PP(i) could be generated from UTP and glucose 1-phosphate through catalysis of UDP-glucose synthase, which provides UDP-glucose, a precursor of glycogen.