Rhodobacter sphaeroides O.U. 001 is a purple non-sulfur bacterium which evolves hydrogen from the breakdown of organic acids under illumination and anaerobic conditions. In this study, the effect of light intensity, light wavelength and illumination protocol on the growth and hydrogen production of R. sphaeroides O.U. 001 was investigated in gas-tight glass photobioreactors with defined medium. The results showed Also it was found that the rate of hydrogen production increased with increasing light intensity and reached saturation at around 270W/m(2) that lack of infrared light (750-950 nm wavelength) decreased photoproduction of hydrogen by 39%. Another factor evaluated was the effect of different illumination protocols on the growth and hydrogen production. It was observed that illumination after inoculation stimulates hydrogen production, increases substrate conversion efficiency and hydrogen production rate; no hydrogen was produced during the dark periods. (c) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.