A pilot-scale study of biohydrogen production was performed in a continuous flow anaerobic fermentation reactor (with an available volume of 1.48 m(3)) for over 200 days. The hydrogen bio-producing reactor (HBR) system was operated under the organic loading rates (OLR) of 3.11-85.57 kg COD/m(3) reactor/d (COD: chemical oxygen demand) with molasses as the substrate. Both biogas and hydrogen yields increased with OLR at the range of 3.11-68.21 kgCOD/m(3) d, but decreased at high OLR (68.21-85.57 kg COD/m(3) reactor/d). The biogas was mainly composed of CO2 and H-2 with the percentage of H-2 ranging from 40% to 52% in biogas. A maximum hydrogen production rate of 5.57 m(3) H-2/m(3) reactor/d, with a specific hydrogen production rate of 0.75 m(3) H-2/kg MLVSS/d, was obtained in the reactor. The hydrogen yield reached 26.13 mol/ka CODremoved within OLR range of 35-55 kg COD/m(3)reactor/d. In addition, it was found that the hydrogen yield was affected by the presence of ethanol and acetate in the liquid phase, and the maximum hydrogen production rate occurred while the ratio of ethanol to acetate was close to 1, due to the adjustment of NAD+/(NADH + H+) through fermentation pathways. Ethanol-type fermentation was favorable for hydrogen production. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.