The potential of using N-2-fixing cyanobacteria to produce hydrogen photobiologically has stimulated research on the physiology and biotechnology of species exhibiting high H-2 production rates over long periods of time. In this work Nostoc flagelliforme, a terrestrial N-2-fixing cyanobacterium, has been examined to establish its physiology and potential for H-2 production under controlled conditions. Cell filaments of N. flagelliforme were purified and grown in liquid culture to optimize its H-2 metabolism. In batch-grown cultures the activity of nitrogenase, the key enzyme for H-2 production in N-2-fixing organisms, was found to be high only during a short phase of exponential growth. A chemostat system was thus constructed for long-term experiments using continuous cultures, with the aim of exploiting the exponential growth phase. The dilution rate (D) and environmental factors, such as N-2 concentration in the gas phase and temperature, significantly influenced H-2 production. Cells grown continuously under the optimized conditions of D = 0.022 h(-1), 34 degrees C and 5.1 kPa N-2 in the gas phase exhibited H-2 production rates that were more than four times higher than the maximal rates under standard batch growth conditions.