Lignocellulosic biomass contains 70-80% carbohydrates and could serve as the ideal feedstock for fermentative hydrogen production. We conducted the pretreatment of corn stover using a steam-explosion process and studied its fermentability for hydrogen production. Using natural inoculant obtained from the heated sludge of a local wastewater treatment plant, we demonstrated that the indigenous microbes were capable of efficiently fermenting the aqueous hydrolyzate derived from the hemicellulose fraction of the steam-pretreated corn stover with and without acid during pretreatment. Biogas contained equal amounts of hydrogen and carbon dioxide. The carbon mass balance is approximately 84%, with acetic and butyric acids as the major carbon byproducts along with carbon dioxide. Hydrogen molar yields of 2.84 and 3.0 were obtained using the mixed sugars present in the hydrolyzate derived from neutral and acidic steam explosion, respectively. These findings verify that hemicellulose from corn stover could be a suitable feedstock for hydrogen production via dark fermentation. (C) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.