In the present work, a combined (acid + thermal) pretreatment was applied for the enhanced fermentative H(2) production (FHP) from Laminaria japonica. Various pretreatment conditions including HCl concentrations, heating temperatures, and reaction times were optimized via response surface methodology (RSM) with a Box-Behnken design (BBD). Through regression analysis, it was found that H(2) yield was well fitted by a quadratic polynomial equation (R(2) = 0.97), and the HCl concentration was the most significant factor influencing FHP. The desirable pretreatment conditions found were HCl concentration of 4.8%, temperature of 93 degrees C, and reaction time of 23 min, under which H(2) yield reached to 159.6 mL H(2)/g dry cell weight (dcw). The main organic acids produced were acetic and butyric acids that related closely with H(2) production. The concentration of hydroxymethylfurfural (HMF), a byproduct formed during the pretreatment process, showed an inverse relationship with H(2) yield, indicating that pretreatment conditions for the H(2) production from L. japonica were successfully optimized, by increasing the hydrolysis rate of the feedstock and also reducing the formation of HMF. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.