In this work, the effects of different parameters on co-culture hydrogen production using Clostridium Butyricum and immobilized Rhodopseudomonas faecalis RLD-53 were investigated. The maximum hydrogen yield of 4.134 mol H(2)/mol glucose was obtained using 6 g/l glucose, 50 mmol/l phosphate buffer, initial pH of 7.5, the ratio of dark to photo bacteria of 1:10 and light intensity of 8000 lux. The maximum hydrogen production rate was 33.85 ml H(2)/l/h. Phosphate buffer concentration was the most important parameter influencing hydrogen production in this co-culture. The ratio of acetate to butyrate increased from 0.74 to 1.82 in the soluble metabolites from C. butyricum with phosphate buffer concentration of 10-50 mmol/l. Experimental results could be of great significance for further pilot studies of co-culture hydrogen production. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.