Nitrogen-fixingAnabaena cylindrica cells are found to evolve hydrogen in high quantities in the presence of CO plus C2H2. Studies with the inhibitors dichlorophenyldimethylurea (DCMU), disalicylidenepropanediamine (DSPD), dibromothymoquinone (DBMIB), undecylbenzimidazole (UDB) and chloro-carbonyl-cyanide-phenylhydrazone (CCCP) and also withAnabaena grown on nitrate- and ammonia-nitrogen show that the H2-formation is due to the ATP-dependent H3O(+)-reduction catalysed by nitrogenase. In control experiments CO plus C2H2 inhibited the activities of a cell-free hydrogenase fromClostridium pasteurianum. It is concluded that Anabaena has a hydrogenase whose natural function is to recycle the H2 lost by the action of nitrogenase.