The sustained production of H-2 by Chlorella pyrenoidosa was achieved without sulfur deficiency or PSII inhibition. C. pyrenoidosa preserved hydrogenase activity for several hours in the dark. Hydrogenase activity in vitro is O-2 sensitive, which indicates that respiration may play an important role in H-2 production. A sustainable production of H-2 was obtained for 200 h under illumination. Further, by using 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a PSII inhibitor, at least 70% electrons for H-2 production were generated from PSII-catalyzed-H2O oxidation. The remaining electrons were probably from endogenous substrate degradation.