Biological technologies for the renewable conversion of biomass to hydrogen, an important chemical feedstock and potential fuel, could reduce greenhouse gas emissions by displacing the current source of virtually all hydrogen: fossil fuels. However, current fermentative methodologies suffer from low productivities and conversion yields. Previous work has shown that purified enzymes from the pentose phosphate pathway can be used to efficiently transfer the reducing equivalents in glucose to NADPH; this approach used a [NiFe] hydrogenase to directly produce hydrogen from NADPH. However, [FeFe] hydrogenases offer much higher hydrogen production activities. We demonstrate a new enzymatic method for the conversion of NADPH reducing equivalents to hydrogen by first using ferredoxin-NADPH-reductase to transfer electrons from NADPH to ferredoxin. The reduced ferredoxin then delivers the electrons to a [FeFe] hydrogenase for hydrogen production. This alternative in vitro pathway enables utilization of the fastest known hydrogenases, [FeFe] hydrogeneases, and activates electron delivery by the native electron donor for these hydrogenases, ferredoxin. We report proof-of-principle data for this synthetic enzyme pathway, showing high volumetric production rates and hydrogenase turnover numbers relative to previous results utilizing [NiFe] hydrogenases. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.