The hydrogenase of Desulfovibrio gigas has been shown to contain one nickel atom, a cluster with three irons and two clusters of the [4Fe-4S] type in an 89 kDa molecule. Though evidence that the nickel ion is involved in the site of hydrogen activation has been presented for this and other hydrogenases, the role of nickel and of the other redox centres in the protein remains to be firmly identified. We have examined the effects of inhibitors of hydrogenase activity in an attempt to identify the functions of the prosthetic redox centres. We have shown carbon monoxide to inhibit at the site of hydrogen activation. The dye, procion red, was found to compete with electron acceptors at a different site, and partial denaturation with the detergent lithium dodecyl sulphate resulted in the differential inhibition of hydrogen activation and substrate reduction. These results imply the presence of distinct domains within the protein with different catalytic activities.