A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O-2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence Of O-2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H+/H-2 redox couple, and H-2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O-2 vary greatly; the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O-2, surviving only if exposed to O-2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O-2 even during turnover and continues to catalyze H-2 oxidation in the presence Of O-2.