Although in theory the amount of hydrogen that could be generated from renewable sources of energy such as cellulose (a polymer of glucose) is vast1, only 16–24% of the maximum stoichiometric yield of hydrogen from glucose (about 12 mol H2 per mol glucose) is typically achieved by biological methods2. Here we show that the enzymes of the oxidative pentose phosphate cycle3,4,5 can be coupled to hydrogenase purified from the bacterium Pyrococcus furiosus, one of only a few hydrogenases that use NADP+ as the electron carrier6, to generate 11.6 mol H2 per mol glucose-6-phosphate. Hydrogen produced by this pathway is the major product, unlike that produced by intermediate metabolic pathways of bacterial fermentation, and therefore has important practical implications for biohydrogen production7.