The precipitous decline in the rates of enzymatic hydrolysis of cellulose with conversion is one of the major limitations to the commercialization of second-generation biofuel. In this work, various rate-limiting factors (fractal kinetics, changes in crystallinity, accessibility, reactivity and hydrolysable fraction, enzyme clogging, and degree of polymerization) were investigated employing experimental as well as computational studies. Model-guided experiments showed cellulose accessibility and the hydrolysable fraction of accessible substrate (a previously undefined and unreported quantity) to decrease steadily until a conversion level of nearly 70%, while cellulose reactivity, defined in terms of hydrolytic activity per amount of actively adsorbed cellulase, remained constant. Substrate depletion, accessibility and hydrolysability decrease accounted for approximately 90% of rate retardation up to 70% conversion. Faster restart rates were observed on partially converted cellulose as compared to uninterrupted hydrolysis rates, supporting an enzyme clogging phenomenon that could possibly be responsible for the additional rate decrease. (C) 2011 Elsevier Ltd. All rights reserved.