A key element in the utilization of lignocellulosic biomass for the production of ethanol is the complete conversion of cellulose and hemicelluloses into simple sugars by cellulases. In general, pretreatments are designed to enhance the accessibility of cellulose microfibrils to enzymes allowing for more efficient conversion. Effective pretreatments loosen, often by selective removal of components, the plant cell wall matrix enabling better penetration and digestion of the wall. It is thought, however, that excessive removal of xylan and lignin may cause the remaining cellulose to collapse into an even more recalcitrant state. In this study, we have monitored how well the major cellulases in a commercial enzyme preparation (Spezyme CP) penetrated the cell wall matrix following dilute acid pretreatments at 100°C, 120°C, and 150°C. Antibodies to the enzymes cel7A (CBHI), cel6A (CBHII), and cel7B (EGI), as well as antibodies to the cell wall matrix components xylan and lignin were detected using immuno-electron microscopy. Dilute acid pretreatment for 20 minutes at 100°C enabled <1% of the thickness of the cell wall to be penetrated by enzyme, pretreatment at 120°C allowed the enzymes to penetrate ~20% of the cell wall, and pretreatment at 150°C allowed 100% penetration of even thickest cell walls. When correlated with cellulose conversion data, these data allow visualization of the dramatic effect pretreatment has on altering the condensed ultrastructure of biomass cell walls. High-resolution localization of enzyme penetration into pretreated biomass is providing insight into the mechanism of action of cellulolytic enzymes on whole, intact biomass cell walls.