Rhodobacter sphaeroides O.U.001 was grown in media with different concentrations of molybdenum and iron to study the expression level of nifD and nifK genes coding for the large subunit of Mo-nitrogenase and hupS gene coding for the small subunit of uptake hydrogenase. Hydrogen productions under the same growth conditions were also evaluated. Increasing concentrations of the molybdenum and iron stimulated hydrogen production and the highest total hydrogen accumulation was achieved in 16.51 mu M sodium molybdare (0.841 H-2/l culture at t =168 h) and 0.1 mM ferric citrate (1.141 H-2/l culture at t =240 h) containing media. Maximal expressions of nifD and nifK were observed from the late log phase until the mid-stationary phase of growth and hupS expression was seen as soon as the hydrogen was produced in the cells. Moreover, nifK, nifD and hupS expressions were significantly reduced in the molybdenum and iron starved cells. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.