Lyngbya majuscula CCAP 1446/4 is a N-2-fixing filamentous nonheterocystous cyanobacterium that possesses two NiFe hydrogenases: an uptake and a bidirectional hydrogenase. The biosynthesis/maturation of NiFe hydrogenases is a highly complex process requiring several proteins. This work presents the characterization of the hyp cluster in L majuscula. The hyp genes are located ca. 8 kb upstream from the uptake hydrogenase structural genes, and seem to be transcribed as a single operon, including three other ORFs interspersed throughout the cluster. In addition, the transcriptional start point was identified 21 bp upstream of hypF, and regulatory sequences were recognized within the promoter region. Several ORFs could also be discerned between the hup and the hyp genes. Moreover, hupW, the gene encoding the putative uptake hydrogenase C-terminal endopeptidase was found 1102 bp downstream of hupL. The hydrogenases maturation process and its regulation are discussed for cyanobacteria possessing both or just one hydrogenase. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.