Powdered wheat solution was fermented batchwise for bio-hydrogen production using heat-treated anaerobic-acidogenic sludge as the inoculum at initial pH of 7.0 and temperature of 37 degrees C. In order to determine the most suitable wheat powder (WP) and cell concentrations yielding the highest hydrogen formation, initial WP concentration was varied between 5 and 30 g L(-1) at a constant initial biomass concentration (2.6 g L(-1)). The initial biomass concentration was varied between 0.5 and 5.0 gL(-1) at constant WP concentration of 20 gL(-1) in another set of experiments. Cumulative bio-hydrogen formation, hydrogen yield and the formation rate were maximum at the WP concentration of 20 g L(-1). Higher WP concentrations resulted in lower fermentation performance probably due to substrate and product (VFA) inhibition. Similarly, hydrogen formation rate and the yield were maximum with a biomass concentration of 2.5 gL(-1). Higher biomass concentrations resulted in lower hydrogen yield and formation rates due to product inhibition. The optimum initial biomass/substrate ratio maximizing the hydrogen yield and formation rate was found to be 0.125 g biomass g(-1) WP. A kinetic model was developed for bio-hydrogen formation from powdered wheat starch and the constants were determined by regression analysis. 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.