We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); hit I Hydrogen Energy 3:1574-1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre-grown under a low light regime (25 mu Em(-2) s(-1)). We failed to establish sustained hydrogen production from Cultures pre-grown under high light (100 mu Em(2) s(-1)). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in Cultures pre-grown under high light. Results showed that light higher than 175 mu Em(-2) s(-1) during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of all artificial electron acceptor (DCBQ) and all inhibitor of electron transport (DBMIB) did not change in cultures pre-grown under 20 mu Em(-2) s(-1) and incubated under 150 mu Em(-2)s(-1) during Sulfur deprivation. In contrast, the potential PSII activity decreased ill Cultures pre-grown under 100 mu Em(-2)s(-1) and incubated under 420 mu Em(-2)s(-1). This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved 0, content during the oxygen evolving stage Of Sulfur deprivation has a negative regulatory role oil PSII activity. To increase hydrogen production by C. reinhardtii pre-grown under 100 mu Em(-2)s(-1), Cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These Cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H-2. It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures.