In this study, optimal basal medium composition for fermentative hydrogen production from cheese whey wastewater was investigated in batch tests. Twenty-five different basal medium formulas was prepared, each containing trace metals concentration (Co, Ni, Zn: 0-5 mg/l), macro elements concentration (Mg: 0-200 mg/l; Mn: 0-10 mg/l, Fe: 0-100 mg/l, Ca: 0-1000 mg/l), C/N ratio of 5-50, and yeast extract and l-cycteine concentrations (0-500 mg/l). Each of the experimental setups was conducted as triplicates in 100-ml serum vials at pH 5.5 under mesophilic conditions (36 +/- 1 degrees C). Granular biomass obtained from an industrial scale upflow anaerobic sludge bed bioreactor was used as an initial inoculum. Considering the hydrogen production efficiencies obtained in this study (max. 3.5 mol H-2/mol lactose(consumed) and 218.6 ml H-2/g lactose(consumed)), a successful nutrient formulation for optimum hydrogen production from cheese whey was proposed as follows; CoCl2, NiCl2, ZnCl2: 1.25-2.5 mg/l; CaCl2: 250-500 mg/l: MgCl2: 50-150 mg/l; MnCl2: 2.5-5 mg/l; FeCl2: 50-100 mg/l; yeast extract: 125-250 mg/l; L-cysteine: 125-250 mg/l and C/N ratio: 30-40.