Clostridium butyricum EB6 successfully produced hydrogen gas from palm oil mill effluent (POME). In this study, central composite design and response surface methodology were applied to determine the optimum conditions for hydrogen production (P(c)) and maximum hydrogen production rate (R(max)) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the hydrogen production and production rate, both individually and interactively. The optimum conditions for hydrogen production (P(c)) were pH 5.69, 36 degrees C, and 92 g COD/l; with an estimated P(c) value of 306 ml H(2)/g carbohydrate. The optimum conditions for maximum hydrogen production rate (R(max)) were pH 6.52, 41 degrees C and 60 g COD/l; with an estimated R(max) value of 914 ml H(2)/h. An overlay study was performed to obtain an overall model optimization. The optimized conditions for the overall model were pH 6.05, 36 degrees C and 94 g COD/l. The hydrogen content in the biogas produced ranged from 60% to 75%. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.