Wheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H-2), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H-2 usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H-2 production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H-2 production from untreated wheat straw ranged from 5.18 to 10.52 mL-H-2 g-VS-1. Whatever the experimental enzyme addition procedure, a two-fold increase in H-2 production yields ranging from 11.06 to 19.63 mL-H-2 g-VS-1 was observed after enzymatic treatment of the wheat straw. The high variability in H-2 yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H-2 production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw(-1). Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.