Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodgepople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto cellulose surface of the set of pretreated substrates were also carried out using an in situ UV–Vis spectrophotometric technique. The cellulose accessibilities measured by the solute exclusion and a cellulose-binding module (CBM)-containing green fluorescent protein (TGC) adsorption methods correlate well for both sets of samples. The substrate enzymatic digestibilities (SEDs) of the hornified substrates are proportional to the measured cellulose accessibilities. Approximately over 90% of the SED was contributed by the accessible pore surfaces of the hornified substrates, suggesting that the substrate external surface plays a minor role contributing to cellulose accessibility and SED. The cellulose accessibilities of the pretreated substrates correlated well with the amounts of cellulase adsorbed. The SEDs of these substrates directly correlated with the amounts of adsorbed cellulase. Biotechnol. Bioeng. 2012; 109:381–389. © 2011 Wiley Periodicals, Inc.