Growth, end-product synthesis, and enzyme activities involved in pyruvate catabolism, H2 synthesis, and ethanol production were studied in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose, pyruvate, glycerol, and combinations of these substrates. Cells could not use pyruvate or glycerol as a sole carbon source, but did metabolize pyruvate in the presence of cellobiose resulting in increased acetate, CO2, H2, lactate and biomass production, and a marginal decrease in formate and ethanol production with increasing pyruvate concentrations (40-120 mM). Comparable concentrations of NaCl did not have an impact on generation time or end product ratios in cellobiose grown cells, eliminating the possibility of a sodium pyruvate induced salt effect. Activities of enzymes involved in pyruvate catabolism and ethanol synthesis did not change, while methyl viologen, ferredoxin, and NAD+-dependant hydrogenase activities increased 10-fold and NADP+-dependant hydrogenase increased 5-fold. The presence of glycerol had no effect on growth or end product synthesis in cellobiose grown cells. End product profiles suggest that carbon from additional intracellular pyruvate flows through pyruvate:ferredoxin oxidoreductase leading to hydrogen and ATP synthesis pathways, and not through pyruvate:formate lyase and NAD(P)H reoxidizing pathways yielding ethanol. The presence of lactate under elevated pyruvate concentrations may be a consequence of buildup of glycolysis intermediates, including fructose-1,6-bisphosphate, an allosteric activator of lactate dehydrogenase, resulting in an alternative method of NADH reoxidation. We demonstrate here that the redox state of a substrate may have important implications on final end product profiles.