Studies have been carried out to establish the ability of O-2-tolerant membrane-bound [NiFe] hydrogenases (MBH) from Ralstonia sp. to catalyze H-2 production in addition to H-2 oxidation. These hydrogenases are not noted for H-2-evolution activity, and this is partly due to strong product inhibition. However, when adsorbed on a rotating disk graphite electrode the enzymes produce H-2 efficiently, provided the H-2 product is continuously removed by rapidly rotating the electrode and flowing N-2 through the gastight electrochemical cell. Electrocatalytic H-2 production proceeds with minimal overpotential - a significant observation because lowering the overpotential (the electrochemically responsive activation barrier) is seen as crucial in developing small-molecule catalysts for H-2 production. A mutant having a high Km for H-2 oxidation did not prove to be a better H-2 producer relative to the wild type, thus suggesting that weak binding of H-2 does not itself confer a tendency to be a H-2 producer. Inhibition by H-2 is much stronger than inhibition by CO and, most significantly, even O-2. Consequently, H-2 can be produced sustainably in the presence of O2 as long as the H-2 is removed continuously, thereby proving the feasibility for biological H-2 production in air.