Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.