Unbeaten, beaten, and recycled softwood pulps were,ere used as substrates to assess the various substrate characteristics that may influence the enzymatic hydrolysis of cellulose. After 4 h of hydrolysis, about 30% of the beaten pulp was hydrolyzed to glucose, as compared with 20% of both unbeaten and recycled pulps, regardless of their similarities in initial fiber length and degree of polymerization. The fragmentation profile (change in fiber length distribution) of both unbeaten and recycled pulps during enzymatic hydrolysis also suggested that they were degraded in a similar fashion. It appeared that the primary action of refining was to enhance swelling of the fibers, resulting in a better fiber-to-fiber contact area, paper strength, and accessibility to cellulase enzymes. In contrast, scanning electron microscopy of partially hydrolyzed residues indicated that recycling caused the disappearance of external fibrilation in the fibers, restoring the fine structure of the pulp to their original unbeaten state. The structural reorganization triggered by recycling also appeared to decrease substrate accessibility to cellulases as well as the paper-making qualities of the fiber. Although substrate characteristics such as crystallinity, particle size, and the degree of polymerization all influence the efficiency of enzymatic hydrolysis, it is primarily the extent of the cellulose surface area available to the enzymes that dictates the rate and degree of hydrolysis of the substrate.