The temperature, initial pH, and substrate concentration on fermentative hydrogen production was optimized by the Box-Behnken design using gamma-irradiated sludge as inoculum, and the fermentation process was analyzed at optimal conditions. The experimental results showed that the maximum cumulative hydrogen production was 3000 mL of H-2/L of medium and the hydrogen yield was 1.81 mol of H-2/mol of glucose, at 32.9 degrees C, with an initial pH of 7.92 and a glucose concentration of 17.0 g/L. The highest hydrogen production rate was accompanied by the exponential growth of microorganisms. The mixed cultures underwent the mixed-acid-type fermentation in the first 10 h and then changed to the acetate butyrate pathway until the end of fermentation. The hydrogen yield showed a positive relationship with acetate generation during the fermentation process.