The enzymatic conversion of sugars to hydrogen could be a promising method for alternative fuel production. Maple tree sap is a source of environmental sugar (e.g., sucrose) that has the potential to be converted into hydrogen using the enzymes invertase, glucose dehydrogenase (GDH), hydrogenase, and glucose isomerase (GI) and the cofactor NADP(+)/NADPH. The kinetics of hydrogen production have been studied, and optimal conditions for hydrogen production are described. At low initial sucrose concentrations, in the absence of glucose isomerase, stoichiometric yields of 1 mol of H-2/mol of sucrose were achieved. At higher sucrose concentrations, the yield of hydrogen declined so that at an initial sucrose concentration of 292 mM only 7% yield of hydrogen was obtained. The reason for this low yield was studied and shown not to be caused by enzyme inactivation or a pH drop during the reaction but due to an instability of the cofactor NADP(+). Although gluconic acid inhibited both NADPH production and oxidation by GDH and hydrogenase, respectively, it was not the major cause of NADP(+) instability. Fructose was also shown to be converted to hydrogen if GI was present in the reaction mixture. Also, by starting with sucrose, 1.34 mol of H-2/mol of sucrose was obtained if GI was present in the reaction mixture.