The need of an efficient and well-characterized heterologous expression system of [FeFe]-hydrogenase for the production of O-2-resistant mutants prompted us to explore the use of Escherichia coli as a possible expression system. O-2-resistant hydrogenase mutants could be instrumental when coupling oxygenic photosynthesis with hydrogen bio-production. In general, expression of Desulfovibrio vulgaris Hildenborough active enzyme in E. coli was very modest indicating that the co-expression of the HydE, HydF and HydG maturases with hydrogenase structural genes in this bacterium is not optimal. A 28-fold increase in activity was obtained when these proteins were co-expressed with the Iron-Sulfur Cluster operon, indicating that one of the problems with over-expression is the correct insertion of FeS clusters. However, the measured activity is still about 4000-fold lower than the one measured in the native hydrogenase indicating that additional, so far unidentified factors may be necessary for optimal heterologous expression of [FeFe]-hydrogenase. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.