Sequential fractionation of AFEX-pretreated corn stover extracts was carried out using ultra-centrifugation, ultra-filtration, and solid phase extraction to isolate various classes of pretreatment products to evaluate their inhibitory effect on cellulases. Ultra-centrifugation removed dark brown precipitates that caused no appreciable enzyme inhibition. Ultra-filtration of ultra-centrifuged AFEX-pretreated corn stover extractives using a 10 kDa molecular weight cutoff (MWCO) membrane removed additional high molecular weight components that accounted for 24-28% of the total observed enzyme inhibition while a 3 kDa MWCO membrane removed 60-65%, suggesting significant inhibition is caused by oligomeric materials. Solid phase extraction (SPE) of AFEX-pretreated corn stover extractives after ultra-centrifugation removed 34-43% of the inhibition; ultra-filtration with a 5 kDa membrane removed 44-56% of the inhibition and when this ultra-filtrate was subjected to SPE a total of 69-70% of the inhibition were removed. Mass spectrometry found several phenolic compounds among the hydrophobic inhibition removed by SPE adsorption. (C) 2013 Published by Elsevier Ltd.