Biological hydrogen production by the green alga Chlamydomonas reinhardtii under sulfur-deprived conditions has attracted great interest due to the fundamental and practical importance of the process. The photosynthetic hydrogen production rate is dependent on various factors such as strain type, nutrient composition, temperature, pH, and light intensity. In this study, physicochemical factors affecting biological hydrogen production by C reinhardtii were evaluated with response surface methodology (RSM). First, the maximum specific growth rate of the alga associated with simultaneous changes of ammonium, phosphate, and sulfate concentrations in the culture medium were investigated. The optimum conditions were determined as NH4+ 8.00 MM, PO43- 1.11 mM, and SO42- 0.79 mM in Tris-acetate-phosphate (TAP) medium. The maximum specific growth rate with the optimum nutrient concentrations was 0.0373 h(-1). Then, the hydrogen production rate of C. reinhardtii under sulfur-deprivation conditions was investigated by simultaneously changing two nutrient concentrations and pH in the medium. The maximum hydrogen production was 2.152 mL of H-2 for a 10-mL culture of alga with density of 6 x 106 cells mL(-1) for 96 h under conditions of NH4+ 9.20 mM, PO43- 2.09 mM, and pH 7.00. The obtained hydrogen production rate was approximately 1.55 times higher than that with the typical TAP medium under sulfur deficiency.