H-2 photoproduction, hydrogenase activities and PSII photochemical activities in Chlorella protothecoides under sulfur (S-) or nitrogen (N-) deprivation or simultaneous N-limitation and S-deprivation were studied. C. protothecoides pre-cultured in full nutrient TAP medium containing 7 mM NH4Cl was found to produce a detectable but low level of H-2, once the cells were inoculated either in S-free or N-free medium. However, cells pre-grown in a low concentration of NH4Cl (0.35 and 0.7 mM) generated a large amount of H-2 after transfer to N-limited and S-free medium. The maximal H-2 outputs of similar to 233.7 and similar to 129.1 ml/l were obtained within 100 h in the cultures exposed to S-deprived medium containing 0.35 mM and 0.7 mM NH4Cl, with the average H-2 production rates being similar to 2.19 and similar to 1.37 ml/l/h, respectively. Our studies further indicated that N-limitation resulted in considerable starch accumulation, chlorophyll synthesis reduction, photosynthetic electron transfer block and oxygen evolving complex (OEC) injury, as well as attenuation in PSII oxygenic activity. Significant starch degradation was not observed during the H-2 photoevolution process. Attenuation of PSII O-2 evolution favored a rapid establishment of anaerobiosis for hydrogenase induction. Meanwhile, a constant high level of hydrogenase activities in C. protothecoides exposed to simultaneous N-limitation and S-deprivation were measured. Based on the above results, a possible mechanism of high H-2 photoproduction in C. protothecoides exposed to N-limitation and S-deprivation was discussed. Low net photosynthetic oxygenic rates, together with high hydrogenase activities were thought to contribute to the enhancement of H-2 photoproduction by C. protothecoides. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.