Fireflies (Coleoptera: Lampyridae) emit various types of light that differ among species and populations of the same species. Their lights are assumed to be biological properties that play important ecological and evolutionary roles. Some species in the Lampyridae emit periodic luminescence, the patterns of which are characterized by species-specific intervals. In previous work, it was predicted that the nitric oxide (NO) regulates the oxygen supply required for the bioluminescence reaction of fireflies. Here, the expression of the NO synthase (NOS) mRNA in some fireflies was examined to verify the predictive model of nitric-oxide-mediated flash control in these insects. The expression of the nos gene in the lantern organ was observed not only in nocturnal flashing species but also in diurnal non-flashing species. It was shown that the expression levels of nos were higher in the lantern of Luciola cruciata (Motschulsky) larvae, which that emits continuous light, than in other body parts, although expression in the lantern of the adults, who flash periodically, was not high. Furthermore, there was no significant difference in expression levels among adults of Luciola cruciata characterized by different flashing intervals. The data do not support the model of an NO-mediated flash control mechanism, during which oxygen becomes available for the luciferin-luciferase reaction through NO-mediated inhibition of mitochondrial respiration. It is also indicated that flash patterns do not co-vary with NOS production. However, high nos expression in the larval lantern suggests that NO may play a role in producing continuous light by functioning as a neurotransmitter signal for bioluminescence.