The lignocellulosic material of mushroom farm waste was used as a feedstock owing to its quantity and availability in Taiwan. In the first stage, the lignocellulosic material of mushroom farm waste was converted into reduced sugars using concentrated sulfuric acid via hydrolysis with a one-step and/or a two-step process. The reduced sugars were then recovered by anion exchange resin. In the second stage, reduced sugars from the first stage were used as substrate to test the potential for biohydrogen production in a batch system. The effects of sulfuric acid concentrations, reaction temperature, particle size, and two-step hydrolysis were investigated at the first pretreatment stage (mushroom farm waste hydrolysis). The reduced sugar concentrations and the initial pH were investigated in the second stage (biohydrogen production). It was found that the maximum yield of reduced sugars in hydrolyzate (reduced sugar per soluble cellulose, wt%) from the first-step (40 degrees C for 20 mm with 55.0% acid) and the second-step (40 degrees C for 20 min with 6.9% acid) were 74.49% and 96.79%, respectively. In the second stage, the best hydrogen production yield and the hydrogen production rate were 2.52 mol H(2)/g COD substrate and 4.38 L/L/d, respectively, were obtained initially with a pH 7.0, a temperature of 37 degrees C and initial reduced sugar concentration of 20 g of COD/L. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.