Dairy wastewater was applied as the source of organic carbon in photobiological generation of hydrogen in the presence of Rhodobacter sphaeroides O.U. 001. Experiments were performed as batch tests with concentration of the waste varying from 5 to 40 v/v %, and concentration of inoculum of 0.36 g dry wt/1 under illumination of 9 klx. The highest volumes of photogenerated hydrogen were obtained at concentration of 40 v/v %, but the maximal substrate yield was reached for lower concentrations of the waste (5-10 v/v %). Changes in concentrations of substrates and products were used for determination of kinetic model. An application of constant pH close to 7 allows for use the non-treated waste in photobiological hydrogen generation with good yield. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.