The genetic background and activities of the enzymes involved in H-2 production were investigated from ten distinct H-2 producing cyanobacteria, revealed by a recent screening. All strains are N-2-fixing, filamentous and heterocystous. Southern hybridization revealed that the tested strains possess the genes encoding the conventional nitrogenase (nifHDK1), and lack the alternative nitrogenases. The high H-2 production rate of these strains was shown not to be dependent on the presence of highly active nitrogenase or bidirectional hydrogenase enzymes. Moreover, most of the strains possessed a highly active uptake hydrogenase enzyme. We also examined the structure of the nif and hup operons encoding nitrogenase and uptake hydrogenase enzymes in the Calothrix 336/3 strain, the best H-2 producer in the screening. We concluded that the ability of the cyanobacteria to produce high levels of H-2 is not directly linked to the maximum capacities of the enzymes involved in H-2 production. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.