The anaerobic production of hydrogen in the dark by bidirectional hydrogenase of unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has been studied by utilizing cells prepared in six types of medium in the presence or absence of D-glucose, NO3-, NaCl and NiCl2. Hydrogen production in this study is unrelated to hydrogenase activity. Hydrogen production is found to bear definite relation to the decreases in the amount of intracellular glucan per unit volume of culture when cells are photosynthetically prepared in BG-11 medium or BG-11 medium with NaCl or NiCl2, or the loss of dry cell mass concentration when cells are photosynthetically prepared in BG-11 medium supplemented with D-glucose or BG-11 medium deficient in NO3-. The cells prepared in BG-11 medium with D-glucose for the production of unit volume of hydrogen are found to reduce the volume of photobioreactor for cell preparation by 41.2% from that without D-glucose.