Aims: To examine the effects of the culture age, illuminance intensity and changes in these parameters during activation on hydrogen generation process carried out by purple nonsulfur Rhodobacter sphaeroides bacteria. Methods and Results: The following parameters were determined in all experiments: the amount of hydrogen evolved (measured using gas chromatography), biomass increase as dry mass, pH values and consumption of organic substance as chemical oxygen demand (COD). The medium used in the process of activation and hydrogen generation contained malic acid (15 mmol) and sodium glutamate (2 mmol). The optimum age of bacteria was 12-24 h and the best intensity of illuminance was found to be 5 cd sr m(-2) on activation and 9 cd sr m(-2) on hydrogen generation. These conditions provided hydrogen evolution of 1.39 l l(-1) of the medium with the highest specific hydrogen production of 0.146 l H-2 l(-1) medium h(-1) g(-1) inoculum. An increase in the illuminance intensity resulted in a slight inhibition of the process. Conclusions: The activation stage of bacteria has a significant effect on the parameters of hydrogen photogeneration. The optimization of the activation stages allowed a shortening of the time of hydrogen generation and of the period after which hydrogen evolution starts. Significance and Impact of the Study: An innovative method of bacteria activation before the initiation of the hydrogen generation process has been used to optimize this process. The shortening of the process duration as well as the twice higher hydrogen yield can help in the designing of other systems (including also those operating under solar irradiation) in which R. sphaeroides bacteria are to be applied.