The hupL gene, encoding the uptake hydrogenase large subunit, in Nostoc sp. strain ATCC 29133, a strain lacking a bidirectional hydrogenase, was inactivated by insertional mutagenesis. Recombinant strains were isolated and analysed, and one hupL(-) strain, NHM5, was selected for further study. Cultures of NHM5 were grown under nitrogen-fixing conditions and H-2 evolution under air was observed using an H-2 electrode. (C) 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.