The H2-oxidation, H2-production and H-3H-exchange activities of the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) were almost completely abolished by Hg(II) and the organic mercurials p-chloromercuribenzoate (pCMB) and p-hydroxymercuriphenylsulphonate. The thiol-modifying reagents N-ethylmaleimide, iodoacetate, dithionitrobenzoate and 2-nitro-5-thiocyanobenzoate had no effect on the activities. Kinetic and spectroscopic measurements suggest that inactivation by pCMB involves at least two reactions; a rapid reaction that is reversed by thiols, and a second, slower and irreversible reaction that occurs at high concentrations of the mercurial. The irreversible reaction was associated with loss of visible absorbance, indicative of a disrupted iron sulphur cluster(s). The effects on the H-3H-exchange activity indicate that the reversible modification affects the H2-activating site. Enzyme that had lost activity due to pCMB treatment, or during long-term storage, was reactivated by thiols. This reactivation was followed by a slower irreversible inactivation, as also occurred with native enzyme; the inactivation was O2 dependent and it was partly prevented by catalase, suggesting that H2O2 may be involved.