Biomass is a potential feedstock for fuels and chemicals, but is primarily composed of cellulose, which is resistant to hydrolysis. It has been hypothesized that the microstructure of cellulose plays an important role in the hydrolysis process; however, current cellulose substrates do not have easily controllable microstructure. Here, we show that the microstructure of cellulose can be controlled by electrospinning nonwoven mats of pure cellulose fibers from solution, and that these fibers are a suitable model substrate for the enzymatic hydrolysis of cellulose. The degree of polymerization, degree of crystallinity, and diameter of the fibers can be controlled by varying the binary solvent and processing conditions for eletrospinning. Cellulose with degrees of polymerization 210, 550, and 1140 were electrospun with two different binary solvents. Fibers electrospun from solutions of cellulose in N-methlymorpholine-N-oxide/water at elevated temperature have mid to high crystallinities (similar to 50-80%), whereas solutions of lithium chloride/dimethylacetimide at room temperature give less crystalline (similar to 30%) cellulose fibers. Varying the infusion rate of the solution or the distance between the nozzle and the collector allows for varying the fiber diameters, producing submicron-through micron-scale fibers with superficial surface areas on the order of similar to 10 m(2)/g. We report some preliminary results for hydrolysis of electrospun fibers with cellulase enzymes, and we demonstrate the potential for kinetics studies with electrospun cellulose fibers to provide insight into how the cellulose microstructure affects the rates of enzymatic hydrolysis.