In light-producing cells (photocytes) of the firefly light organ, mitochondria are clustered in the cell periphery, positioned between the tracheolar air supply and the oxygen-requiring bioluminescent reactants which are sequestered in more centrally-localized peroxisomes. This relative positioning suggests that mitochondria could control oxygen availability for the light reaction. We hypothesized that active cellular respiration would make the interior regions of the photocytes relatively hypoxic, and that the “on” signal for production of bioluminescence might depend on inhibition of mitochondrial oxygen consumption, which would allow delivered oxygen to pass through the peripheral mitochondrial zone to reach peroxisomes deep in the cell interior. We published recently that exogenous NO induces bioluminescence in the intact firefly; that NO mediates octopamine-induced bioluminescence in the dissected lantern, and that nitric oxide synthase is abundant in cells of the tracheolar system of the light organ. Additional experiments showed that nitric oxide gas (NO) inhibits respiration in isolated lantern mitochondria. Inhibition is reversed by bright light, and this inhibition is relieved when the light is turned off. Altogether, the results support the idea that NO triggers light production by reversible inhibition of mitochondrial respiration in lantern cells, and probably in tracheolar cells as well. The data also suggest that the light of bioluminescence itself relieves NO inhibition thus contributing to rapid on/off switching. While other mechanisms may be in play, NO production that is directly related to neural input appears to have a key role in the oxygen gating that controls flash communication signals.