Sequential hydrolysis of oat straw and hydrogen (H-2) production from hydrolysates and hydrolysates constituents were investigated. One acid-enzymatic and two acid-alkalineenzymatic procedures were assessed to measure their capability to convert hemicellulose and cellulose from oat straw to sugars. Acid hydrolysis, using HCl, resulted effective to depolymerize hemicellulose (81%) and also facilitated the activity of cellulases over remaining fiber. When alkaline hydrolysis, using KOH/NaClO2/KOH or NaOH/H2O2, were applied, the depolymerization of cellulose was only slightly increased. Total sugar recoveries ranged from 69 to 79% in the different sequential hydrolysis tested. Hence, hydrolysates from sequential acid-enzymatic hydrolysis were used as substrates for H-2 production in batch assays. The enzymatic hydrolysate produced a higher H-2 molar yield (2.39 mol H-2/mol reducing sugars) than the acid hydrolysate (1.1 mol H-2/mol reducing sugars). It was found that lower H-2 production from the acid hydrolysate was partially due to a lower H-2 yield from arabinose and not to measured microbial inhibitors (furfural, hydroxymethylfurfural, vanillin and syringaldehyde). Also, it was found that the commercial enzyme (Celluclast 1.5 L) was easily fermented, and greatly contributed to the H-2 production. This is the first study that provides experimental evidence of H-2 production from fermentation of a commercial enzyme. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.