The green alga, Chlamydomonas reinhardtii, can photoproduce molecular H(2) via ferredoxin and the reversible [Fe]hydrogenase enzyme under anaerobic conditions. Recently, a novel approach for sustained H(2) gas photoproduction was discovered in cell cultures subjected to S-deprived conditions (A. Melis, L. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert [2000] Plant Physiol 122: 127-135). The close relationship between S and Fe in the H(2)-production process is of interest because Fe-S clusters are constituents of both ferredoxin and hydrogenase. In this study, we used Mössbauer spectroscopy to examine both the uptake of Fe by the alga at different CO(2) concentrations during growth and the influence of anaerobiosis on the accumulation of Fe. Algal cells grown in media with (57)Fe(III) at elevated (3%, v/v) CO(2) concentration exhibit elevated levels of Fe and have two comparable pools of the ion: (a) Fe(III) with Mössbauer parameters of quadrupole splitting = 0.65 mm s(-1) and isomeric shift = 0.46 mm s(-1) and (b) Fe(II) with quadrupole splitting = 3.1 mm s(-1) and isomeric shift = 1.36 mm s(-1). Disruption of the cells and use of the specific Fe chelator, bathophenanthroline, have demonstrated that the Fe(II) pool is located inside the cell. The amount of Fe(III) in the cells increases with the age of the algal culture, whereas the amount of Fe(II) remains constant on a chlorophyll basis. Growing the algae under atmospheric CO(2) (limiting) conditions, compared with 3% (v/v) CO(2), resulted in a decrease in the intracellular Fe(II) content by a factor of 3. Incubating C. reinhardtii cells, grown at atmospheric CO(2) for 3 h in the dark under anaerobic conditions, not only induced hydrogenase activity but also increased the Fe(II) content in the cells up to the saturation level observed in cells grown aerobically at high CO(2). This result is novel and suggests a correlation between the amount of Fe(II) cations stored in the cells, the CO(2) concentration, and anaerobiosis. A comparison of Fe-uptake results with a cyanobacterium, yeast, and algae suggests that the intracellular Fe(II) pool in C. reinhardtii may reside in the cell vacuole.