In the production of acetate by Clostridium thermolacticum growing on lactose, considerable amounts of hydrogen were generated. Lactose available in large amounts from milk permeate, a wastestream of the dairy industry, appears to be a valuable substrate for cheap production of biohydrogen. In this study, continuous cultivation of C thermolacticum was carried out in a bioreactor, under anaerobic thermophilic conditions, on minimal medium containing 10 g l(-1) lactose. Different dilution rates and pH were tested. C thermolacticum growing on lactose produced acetate, ethanol and lactate in the liquid phase. For all conditions tested, hydrogen was the main product in the gas phase. Hydrogen specific production higher than 5 mmol H-2 (g cell)(-1) h(-1) was obtained. By operating this fermentation at high-dilution rate and alkaline pH, the hydrogen content in the gas phase was maximized. (C) 2004 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.