The role of different chemical and physical factors in enhancing biohydrogen production from xylose using a mixed anaerobic culture was examined under mesophilic conditions. A fractional factorial design (FFD) 3((k-p)) was used to optimize pH, the oleic acid (OA) concentration and the biomass concentration. The FFD analysis indicated that the hydrogen (H-2) yield was affected by 3 single factors as well as by 2 factor interactions. Under optimum conditions (1600 mg L-1 of oleic acid (OA) and 1900 mg L-1 VSS and pH 6.7), the H-2 yield reached 2.64 +/- 0.12 mol mol(-1) of xylose (80% of the theoretical yield). Based on the ANOVA and Pareto chart analysis, the linear and quadratic OA and pH terms were significant and the linear and quadratic VSS terms were insignificant. Normally distribution of the residuals was confirmed from the Anderson-Darling (AD) plot. The studentized residuals versus the predicted values plot clearly demonstrated that the data points were randomly scattered. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.