The effect of ferrous ion (0-3.2 mg/l) on photo heterotrophic hydrogen production was studied in batch culture using sodium lactate as substrate. The results showed that hydrogen production by Rhodobacter sphaeroides was significantly suppressed when Fe2+ was limited. Hydrogen production increased linearly with an increase in Fe2+ concentration in the range of 0-1.6 mg/l; reaching a maximum at 2.4 mg/l. When hydrogen production was suppressed in the above medium, a pH increase to 8.9 was observed, and the ratio of lactate utilized to total organic carbon removal was found to be increased, indicating that more soluble organic products were produced. Under the Fe2+ limited conditions, ferrous iron was shown to have a greater effect on hydrogen production by Rb. sphaeroides than that by the anaerobic heterotrophic bacterium Clostridium butyricum. (C) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.