The present study examines the feasibility of producing hydrogen by dark fermentation using unsterilised cheese whey in a UASB reactor. A lab-scale UASB reactor was operated for more than 250 days and unsterilised whey was used as the feed. The evolution of the microbial community was studied during reactor operation using molecular biology tools (T-RFLP, 16S rRNA cloning library and FISH) and conventional microbiological techniques. The results showed that hydrogen can be produced but in low amounts. For the highest loading rate tested (20 gCOD/L.d), hydrogen production was 122 mL H(2)/L.d. Maintenance of low pH (mean = 5) was insufficient to control methanogenesis; methane was produced concomitantly with hydrogen, suggesting that the methanogenic biomass adapted to the low pH conditions. increasing the loading rate to values of 2.5 gCOD/gVSS.d favoured hydrogen production in the reactor. Microbiological studies showed the prevalence of fermentative organisms from the genera Megasphaera, Anaerotruncus, Pectinatus and Lactobacillus, which may be responsible for hydrogen production. However, the persistence of methanogenesis and the presence of other fermenters, not clearly recognised as hydrogen producers indicates that competition for the substrate may explain the low hydrogen production. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.