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X-ray crystallographic studies [Ogata et al., J. Am. Chem. Soc. 124 (2002) 11628–11635] have shown that carbon monoxide binds to the nickel ion at the active site of the [NiFe] hydrogenase from Desulfovibriovulgaris Miyazaki F and inhibits its catalytic function. In the present work spectroscopic aspects of the CO inhibition for this bacterial organism are reported for the first time and enable a direct comparison with the existing crystallographic data. The binding affinity of each specific redox state for CO is probed by FTIR spectro-electrochemistry. It is shown that only the physiological state Ni–SIa reacts with CO. The CO-inhibited product state is EPR-silent (Ni2+) and exists in two forms, Ni–SCO and Ni–SCOred. At very negative potentials, the exogenous CO is electrochemically detached from the active site and the active Ni–R states are obtained. At temperatures below 100 K, photodissociation of the extrinsic CO from the Ni–SCO state results in Ni–SIa that is identified to be the only light-induced state. In the dark, rebinding of CO takes place; the recombination rate constants are of biexponential character and the activation barrier is determined to be approximately 9 kJ mol−1. In addition, formation of a paramagnetic CO-inhibited state (Ni–CO) was observed that results from the interaction of carbon monoxide with the Ni–L state. It is proposed that the nickel in Ni–CO is in a formal monovalent state (Ni1+).