We demonstrated that a significant volume of H-2 gas could be photobiologically produced by a marine green alga Platymonas subcordiformis when an uncoupler of photophosphorylation, carbonyl cyanide in-chlorophenylhydrazone (CCCP), was added after 32 h of anaerobic dark incubation, whereas a negligible volume of H-2 gas was produced without CCCP. The role of CCCP in enhancing photobiological H-2 production was delineated. CCCP as an ADRY agent (agent accelerating the deactivation reactions of water-splitting enzyme system Y) rapidly inhibited the photosystem H (PSII) activity of P. subcordiformis cells, resulting in a markedly decline in the coupled oxygen evolution. The mitochondrial oxidative respiration was only slightly inactivated by CCCP, which depleted O-2 in the light. As a result, anaerobiosis during the stage of photobiological H-2 evolution was established, preventing severe O-2 inactivation of the reversible hydrogenase in P. subcordiformis. The uncoupling effect of CCCP accelerates electron transfer from water due to a disruption of the proton motive force and release of Delta pH across the thylakoid membrane and thus enhances the accessibility of electron and H+ to hydrogenase. The electrons for hydrogen photoevolution are mainly from the photolysis of water (90%). Upon the addition of CCCP, Chl a/b ratio increased, which implies a decrease in the light-harvesting PSII antennae or an increase in PSII/PSI ratio, possibly resulting in higher efficiency of utilization of light energy. The enhancement of H-2 evolution by the addition of CCCP is mostly due to the combination of the above three mechanisms. However, the disruption of the proton gradient across the thylakoid membrane may prevent a sustained photobiological H-2 evolution due to a shortfall of ATP generation essential for the maintenance and repair functions of the cells.