The genetic background and activities of the enzymes involved in H2 production were investigated from ten distinct H2 producing cyanobacteria, revealed by a recent screening. All strains are N2-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 H2 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 H2 producer in the screening. We concluded that the ability of the cyanobacteria to produce high levels of H2 is not directly linked to the maximum capacities of the enzymes involved in H2 production.