Brewer's spent grain (BSG) is a promising feedstock for ABE fermentation. Sulfuric acid pretreatment of BSG at pH 1, 121 degrees C and different solid loadings (5-15% w/w) was investigated. Enzymatic hydrolysis and ABE fermentation by Clostridium beijerinckii DSM 6422 of non-washed and washed pretreated BSG were performed to compare monosaccharide release and butanol production. Pretreatment at 15% w/w BSG resulted in higher availability of sugars in both the enzymatic hydrolysates and pretreatment liquid, and overall yields of 75 g butanol/kg BSG and 95 g ABE/kg BSG were obtained. When the enzymatic hydrolysate from the washed pretreated BSG was fermented, butanol (6.0 +/- 0.5 g/L) and ABE (7.4 +/- 1.0 g/L) concentrations were lower compared with 7.5 +/- 0.6 g/L butanol and 10.0 +/- 0.8 g/L ABE from a control. The fermentation of the liquid released in the pretreatment at 15% w/w resulted in a butanol production of 6.6 +/- 0.8 g/L with a total ABE of 8.6 +/- 1.3 g/L after overliming.