Occurrence and localization of an uptake hydrogenase were examined in three strains of the blue-green alga, Anabaena. In vivo H(2) uptake was detected (0.60-1.44 mumoles/[mg of chlorophyll a per hour]) in all three strains when grown with N(2) as the sole source of nitrogen. H(2) uptake (in vivo and in vitro) was severely suppressed in cultures grown on NH(4) (+) and lacking heterocysts. H(2) uptake in cell-free extracts could be readily measured with a methyl viologen-ferricyanide electron acceptor system. Solubilization kinetics during cavitation of aerobically grown Anabaena 7120 indicates that the uptake hydrogenase is localized solely in the heterocyst. When the same organism is grown on N(2)/CO(2), vegetative cells may account for up to 21% of the total hydrogenase activity in the filaments. The results are discussed in terms of a proposed functional relationship between nitrogenase and hydrogenase.