We have discovered a new competitive pathway for O-2 sensitivity in algal H-2 production that is distinct from the O-2 sensitivity of hydrogenase per se. This O-2 sensitivity is apparently linked to the photosynthetic H-2 production pathway that is coupled to proton translocation across the thylakoid membrane. Addition of the proton uncoupler carbonyl cyanide-p-trifluoro-methoxy-phenylhydrazone eliminates this mode Of O-2 inhibition on H-2 photoevolution. This newly discovered inhibition is most likely owing to background O-2 that apparently serves as a terminal electron acceptor in competition with the H-2 production pathway for photosynthetically generated electrons from water splitting. This O-2-sensitive H-2 production electron transport pathway was inhibited by 3[3,4-dichlorophenyl]1,1-dimethylurea. Our experiments demonstrated that this new pathway is more sensitive to O-2 than the traditionally known O-2 sensitivity of hydrogenase. This discovery provides new insight into the mechanism of O-2 inactivation of hydrogenase and may contribute to the development of a more-efficient and robust system for photosynthetic H-2 production.