The reaction between hydrogen and the [NiFe]-hydrogenase from Allochromatium vinosum in its inactive form has been studied by stopped-flow infrared spectroscopy. The data, for the first time, clearly show that at room temperature enzyme in the unready state, either oxidized or reduced, does not react with hydrogen. Enzyme in the ready state reacts with hydrogen after a lag phase of about six seconds, whereby a specific reduction of the enzyme occurs. The lag phase and the rate of reduction of the ready enzyme are neither dependent on the enzyme concentration nor on the substrate concentration, i.e., substoichiometric and 8-fold excess amounts of H(2) reduce the ready enzyme at the same rate. Oxygen delays this reaction but does not prevent it. The infrared changes lead us to suggest that the hydroxyl group, bridging between the Ni and the Fe atom in the active site, becomes protonated during this reduction. At physiological temperatures, this property of the inactive ready enzyme enables a full development of activity by substoichiometric H(2) concentrations.