Screening of the University of Helsinki Culture Collection for naturally good H2 producing cyanobacteria recently revealed several promising strains. One of the superior strains is Calothrix 336/3, an N2-fixing heterocystous filamentous cyanobacterium. Making use of an important feature of the Calothrix 336/3 cells to adhere to the substrate, we applied an immobilization technique to improve H2 production capacity of this strain. We examined the basic properties of immobilization in Ca2+-alginate films in response to the production of H2 of the Calothrix 336/3 strain and as reference strains we used a model organism Anabaena PCC 7120 and its uptake hydrogenase mutant, ΔhupL, that allow us to compare the responses of different strains to alginate entrapment. Immobilization of the Calothrix 336/3 and ΔhupL mutant cells in Ca2+-alginate resulted in prolonged H2 production over several cycles. Immobilization of the Calothrix 336/3 cells was most successful and production of H2 could be measured even after 40 days after immobilization.