The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady-state O2 concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters Nm, Nd, and α were derived. In both strains, nitrogenase activity (Ntot = Nm + Nd) was the highest at 39°C–42°C and at 0% O2. The ratio Ntot/Nd was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (Nd). Ntot/Nd increased with decreasing O2 concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light-driven nitrogenase activity (Nm). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O2 concentration and temperature as the latter may dynamically alter the flux of O2 into the heterocyst.