ABSTRACT Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PP _i )-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase ( ppdk ) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum . The requirement for PP _i in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PP _i -dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PP _i must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PP _i generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol.