Fenton's reagent that generates reactive hydroxyl radical species was evaluated for its effectiveness as a pretreatment agent on cotton cellulosic substrates to increase its susceptibility to cellulase enzyme. Response surface methodology was used to optimize four different process variables viz., time of reaction; substrate size and concentrations of Fe(2+) and H(2)O(2). Overall, the cellulose substrates treated at 0.5 mM concentration of Fe(2+), 2% concentration of H(2)O(2) for a reaction period of 48 h gave the highest enzyme activity as determined using the response surface methodology. Cellulose substrates with high aspect ratio recorded better enzyme response than that with low aspect ratio which is supported by copper number estimation. The cellulosic substrate prepared using a combination of optimized Fenton's pretreatment conditions and/or enzyme hydrolysis were studied and characterized by atomic force microscopy and scanning electron microscopy. Additionally, degree of polymerization analysis gives further insight into the degradation during Fenton's reaction. (C) 2011 Elsevier Ltd. All rights reserved.