This paper focuses on the production of bio-hydrogen by dark fermentation of de-proteinized (by membrane ultra-filtration) cheese whey, an abundant waste stream of the food industry. This waste stream contains mainly lactose, which was also used as reference substrate in bio-hydrogen production tests. Those tests were conducted in batch by using a thermally treated anaerobic sludge as inoculum offermentative bacteria. Tests were performed under different buffering conditions and initial substrate concentrations. The maximum hydrogen production yield (2.8-3.6 mol H2 mol lactose-1) was obtained when the initial substrate was between 9 and 15 g COD L-1, and by adding an appropriate amount of an organic buffer. This yield is slightly lower than the theoretical one of 4 mol H2 mol lactose-1 which is expected when both acetic and butyric acids are produced. The biogas contained 47 % of H2 and 53 % of CO2. The residual soluble end products were mainly acetic and butyric acid at a molar ratio of approximately 1:1. Significant formic acid concentrations were also observed, whereas no solventogenesis products were detected.