The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H-2 production. H-2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H(2)ase), the key enzyme responsible for H-2 metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H(2)ase by O-2 coevolved in photosynthesis. Recently, we achieved sustainable H-2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H(2)ase. Employing membrane-inlet mass spectrometry and (H2O)-O-18, we now present clear evidence that efficient H-2 photoproduction in pulseilluminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H(2)ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H-2 photoproduction ceases and instead a slow overall H-2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H-2 photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H-2 yield.