In N(2)-fixing cyanobacteria, the reduction of N(2) to NH(3) is coupled with the production of molecular hydrogen, which is rapidly consumed by an uptake hydrogenase, an enzyme that is present in almost all diazotrophic cyanobacteria. The cellular and subcellular localization of the cyanobacterial uptake hydrogenase remains uncertain, and it is definitely strain dependent. Previous studies focused mainly on heterocystous cyanobacteria and used heterologous antisera. The present work represents the first effort to establish the subcellular localization of the uptake hydrogenase in a N(2)-fixing filamentous nonheterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/4, using the first antiserum produced against a cyanobacterial uptake hydrogenase. The data obtained revealed higher specific labelling associated with the thylakoid membranes of L. majuscula, reinforcing the idea that the cyanobacterial uptake hydrogenase is indeed a membrane-bound protein. For comparative purposes, the localization of the uptake hydrogenase was also investigated in two distinct heterocystous cyanobacterial strains, and while in Nostoc sp. PCC 7120 the labelling was only observed in the heterocysts, in Nostoc punctiforme, the presence of uptake hydrogenase antigens was detected in both the vegetative cells and heterocysts, corresponding most probably to an inactive and an active form of the enzyme.