Oat straw was subjected to sequential pretreatment: acid/alkaline/enzymatic, to convert the lignocellulosic material in soluble sugars. The hydrolysates from acid pretreatment (2% HCl, 90 degrees C) and enzymatic pretreatment (cellulase, pH 4.5, 45 degrees C) were used as substrates in two lab-scale UASB reactors for methane production. The acid and enzymatic hydrolysates contained 25.6 and 35.3 g/L of total sugars, respectively, which corresponded to a COD of 23.6 and 30.5 g/L, respectively. The UASB reactor fed with acid hydrolysate achieved a maximum methane yield of 0.34 L CH4/g COD at an organic loading rate (OLR) of 2.5 g COD/L-d. In the reactor fed with enzymatic hydrolysate the methane yield was 0.36 LCH4/g COD at OLR higher than 8.8 g COD/L-d. The anaerobic digestion of both hydrolysates was feasible without the need of a detoxification step. The sequential pretreatment of oat straw allowed to solubilize 96.8% of hemicellulose, 77.2% of cellulose and 42.2% of lignin. (C) 2012 Elsevier Ltd. All rights reserved.