[FeFe] hydrogenases catalyze the rapid combination of protons and electrons into hydrogen, but their oxygen sensitivity limits their use in clean renewable hydrogen production applications. The catalytic activity of current [FeFe] hydrogenases is destroyed by oxygen. Here, the discovery of mutant [FeFe] hydrogenases with decreased oxygen sensitivity is described. The new hydrogenases are derived from [FeFe] hydrogenase I of Clostridium pasteurianum (CpI). A cell-free protein synthesis-based screening platform was used to identify an initial mutant from a randomly mutated CpI library. Three mutations were cooperatively responsible for the decreased oxygen sensitivity, and further improvements were identified by saturation mutagenesis at the influential sites. After oxygen exposure under conditions where the enzyme is in the resting state, the mutant hydrogenase retains significantly higher methyl viologen reduction activity than the wildtype enzyme. However, surprisingly, when the enzyme is actively catalyzing hydrogen production during oxygen exposure, the mutant hydrogenase shows no improved oxygen tolerance. This observation highlights the complexity of the oxygen inactivation process in CpI and demonstrates the need to develop a screen that measures hydrogenase oxygen tolerance during catalysis. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.