Screening of the University of Helsinki Culture Collection for naturally good H-2 producing cyanobacteria recently revealed several promising strains. One of the superior strains is Calothrix 336/3, an N-2-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 H-2 production capacity of this strain. We examined the basic properties of immobilization in Ca2+-alginate films in response to the production of H-2 of the Calothrix 336/3 strain and as reference strains we used a model organism Anabaena PCC 7120 and its uptake hydrogenase mutant, Delta hupL, that allow us to compare the responses of different strains to alginate entrapment. Immobilization of the Calothrix 336/3 and Delta hupL mutant cells in Ca2+-alginate resulted in prolonged H-2 production over several cycles. Immobilization of the Calothrix 336/3 cells was most successful and production of H-2 could be measured even after 40 days after immobilization. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.