This study investigates the effects of temperature and substrate concentration on biological hydrogen production from starch using mixed cultures. In this work, although hydrogen was successfully produced under the thermophilic condition, stable hydrogen production was not observed under the mesophilic condition. In the thermophilic reactor, the maximum hydrogen yield was 2.8 mol H(2)/mol glucose at 20 g/l-starch; however, hydrogen yield decreased drastically with the change of by-product distribution when substrate concentration was over 30 g/l-starch. A negative correlation was observed between the hydrogen yield and the total concentration of undissociated acids. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.