A pretreatment that combines a thermo-mechanical process (extrusion) with chemical and biological catalysts to produce fermentable sugars from barley straw (BS) biomass was investigated. BS was firstly extruded with alkali and then, the pretreated material (extrudate) was submitted to extrusion with hydrolytic enzymes (bioextrusion). The bioextrudate was found to have 35% (w/w dwb) of total solids in soluble form, partly coming from carbohydrate hydrolysis during bioextrusion. About 48% of soluble solids dry weight is comprised by sugars, mostly glucose and xylose. Further enzymatic hydrolysis of bioextrudate could be successfully carried out at high solid loading level of 30% (w/v), with sugar production yield of 32 g glucose and 18 g xylose/100 g bioextrudate at 72 h incubation (equivalent to 96 and 52 g/l concentration, respectively). These results, together with the high level of integration of the process, indicate a great potential of this pretreatment technology for sugar production from lignocellulosic substrates. (C) 2014 Elsevier Ltd. All rights reserved.