Statistical experimental designs were applied for the optimization of medium constituents for hydrogen production from xylose by newly isolated Enterobacter sp. CN1. Using Plackett-Burman design, xylose, FeSO(4) and peptone were identified as significant variables which highly influenced hydrogen production. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. These variables were subsequently optimized using Box-Behnken design of response surface methodology (RSM). The optimum conditions were found to be xylose 16.15 g/L, FeSO(4) 250.17 mg/L, peptone 2.54 g/L. Hydrogen production at these optimum conditions was 1149.9 +/- 65 ml H(2)/L medium. Under different carbon sources condition, the cumulative hydrogen volume were 1217 ml H(2)/L xylose medium, 1102 ml H(2)/L glucose medium and 977 ml H(2)/L sucrose medium; the maximum hydrogen yield were 2.0 +/- 0.05 mol H(2)/mol xylose, 0.64 mol H(2)/mol glucose. Fermentative hydrogen production from xylose by Enterobacter sp. CN1 was superior to glucose and sucrose. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.