It was previously shown that Clostridium acetobutylicum is capable to over-express various [FeFe] hydrogenases although the protein yield was low. In this study we report on doubling the yield of the clostridial hydrogenase by replacing the native gene hydA1(Ca) with a recombinant one via homologous recombination. The purified protein HydA1(Ca) shows an unexpected high specific activity (up to 2257 mu mol H(2) min(-1) mg(-1)) for hydrogen evolution. Furthermore, the highly active green algal hydrogenase HydA1(Cr) from Chlamydomonas reinhardtii was heterologously expressed in C. acetobutylicum, and purified with increased yield (1 mg protein per liter of cells) and high activity (625 mu mol H(2) min(-1) mg(-1)). EPR studies demonstrate intact H-clusters for homologously and heterologously expressed [FeFe] hydrogenases in the CO-inhibited oxidized redox state, and prove the high efficiency of the C. acetobutylicum expression system. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.