Effective nitrogenase synthesis by nostocacean cyanobacteria [Anabaena spiroides, A. varia- bilis and another isolate] (including one mutant strain unable to form heterocysts or fix N aerobically) was induced in the light in the absence of molecular O2. Anaerobiosis was maintained during induction by treating the organisms with dichloromethylurea which prevents photosynthetic O2 production. Under these special conditions both synthesis and activity of nitrogenase were light-dependent, the required ATP being produced by cyclic photophosphorylation. Enzyme synthesis and activity were also dependent on the availability of an organic substrate that could serve both as a general source of C and as a source of reductant. The organic requirement could be fulfilled by the intracellular glycogen reserve or, in facultative heterotrophs, by a utilizable sugar (e.g., glucose). Nitrogenase synthesized anaerobically was highly susceptible to inactivation by molecular O2 in vivo: exposure of a suspension of anaerobically induced filaments to 20% (vol/vol) O2 for 1 h caused total and irreversible destruction of the enzyme. Anaerobic nitrogenase synthesis was not accompanied by the differentiation of mature heterocysts, the morphogenetic process being arrested at an early (proheterocyst) stage. After the gratuitous anaerobic synthesis of nitrogenase, introduction of either N2, nitrate or ammonia to the illuminated, anaerobic suspension resulted in a rapid accumulation of cyanophycin granules in both vegetative cells and proheterocysts. Cyanophycin was randomly deposited in vegetative cells, but localized at the cell poles of the proheterocysts. The bearing of these findings on the role played by the heterocyst in N fixation is discussed.