Sulfate nutrient-deprivation in Chlamydomonas reinhardtii brings about prompt degradation of Rubisco and a concomitant substantial accumulation of starch. These changes precede hydrogen (H-2) photoevolution by the cells. The cause-and-effect relationship between Rubisco loss, starch accumulation and subsequent H-2-photoevolution in C reinhardtii, and the role of illumination for these changes to occur, was investigated in this work. A Rubisco-less and acetate-requiring mutant of C reinhardtii (CC2653) was employed as a tool in this investigation and compared to the wild type (WT) in terms of protein and starch metabolic flux and H-2-evolution upon sulfur deprivation. Results showed a prompt Rubisco degradation and concomitant 10-fold starch accumulation in the WT in the light, which was completed within 48 h of S-deprivation. This was followed by a regulated starch degradation and concomitant H-2-photoevolution, which lasted for up to 120 h in S-deprivation. This massive flux of primary metabolites (protein and starch) did not occur in the dark in the WT, suggesting a strictly light-dependent and integrated process in metabolite rearrangement and H-2-photoevolution in C reinhardtii. The Rubisco-less CC2653 mutant failed to accumulate starch upon S-deprivation in the light or dark and also failed to evolve H2 gas. These results suggested a temporal cause-and-effect relationship between the light-dependent catabolism of Rubisco and starch accumulation, and the subsequent ability of the cell to perform a light-dependent starch degradation and H-2-photoevolution. The regulated starch breakdown in the light apparently provides the endogenous substrate that supports H-2-evolution, both by feeding electrons into the plastoquinone pool in chloroplasts, and indirectly by sustaining mitochondrial respiration for the maintenance of anaerobiosis in the cell. (c) 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.