The Ni-C state of the NiFe hydrogenase from Desulfovibrio gigas was found to be stable for over 40 h at pH 8.0 in the strict absence of H-2. This demonstrates that Ni-C is unable to spontaneously reduce protons to H-2 under these conditions, a result that is contrary to earlier reports. The form of the nickel that spontaneously reduces protons is probably Ni-R, the EPR-silent state that appears to be one electron more reduced than Ni-C, The stability of Ni-C permitted, for the first time, stoichiometric oxidative titrations of the H-2-free, reduced enzyme. Four such titrations were performed, using the oxidant thionin and monitoring the progress of the titrations by electronic absorption (at 410 nm) and EPR spectroscopies. Redox changes in the enzyme’s two Fe4S4 clusters were readily followed by changes at 410 nm, while those of the Ni were observed by EPR. Redox changes of the Fe3S4 cluster were monitored by both spectroscopic methods. At the start of the titrations, the Ni center was in the Ni-C state and the Fe-S clusters were partially oxidized. Adding thionin caused disappearance of Ni-C, development of the EPR-silent intermediate state, and eventually the appearance of Ni-B. The Fe-S clusters oxidized gradually throughout every stage of the titrations. An average of 4.2 oxidizing equiv/mol of thionin was consumed overall. After the number of equiv/mol consumed by the Fe-S clusters and the EPR-active Ni ions were substracted, an average of 1.2 oxidizing equiv/mol remained unassigned. The activity of the enzyme appears to be correlated to the ability of the Ni site to undergo redox chemistry.