In the present study, the possible involvement of reactive oxygen species (ROS) in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis of Bombyx mori prothoracic glands (PGs) was investigated. Results showed that PTTH treatment resulted in a rapidly transient increase in the intracellular ROS concentration, as measured using 2',7'-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant, N-acetylcysteine (NAC), abolished PTTH-induced increase in fluorescence. Furthermore, PTTH-induced ROS production was partially inhibited by the NAD(P)H oxidase inhibitor, apocynin, indicating that NAD(P)H oxidase is one of the sources for PTTH-stimulated ROS production. Four mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenylene iodonium (DPI)) significantly attenuated ROS production induced by PTTH. These data suggest that the activity of complexes I and III in the electron transport chain and the mitochondrial inner membrane potential (ΔΨ) contribute to PTTH-stimulated ROS production. In addition, PTTH-stimulated ecdysteroidogenesis was greatly inhibited by treatment with either NAC or mitochondrial inhibitors (rotenone, antimycin A, FCCP, and DPI), but not with apocynin. These results indicate that mitochondria-derived, but not membrane NAD(P)H oxidase-mediated ROS signaling, is involved in PTTH-stimulated ecdysteroidogenesis of PGs in B. mori.