The fumigant insecticide phosphine (PH₃) is known to inhibit cytochrome c oxidase in vitro. Inhibition of the respiratory chain at this site has been shown to stimulate the generation of superoxide radicals (O₂⁻), which dismutate to form hydrogen peroxide (H₂O₂). This study was performed in order to investigate the production of H₂O₂ by mitochondria isolated from granary weevil (Sitophilus granarius) and mouse liver on exposure to PH₃. Other respiratory inhibitors, antimycin, myxothiazol, and rotenone were used with insect mitochondria. Hydrogen peroxide was measured spectrophotometrically using yeast cytochrome c peroxidase as an indicator. Insect and mouse liver mitochondria, utilizing endogenous substrate, both produced H₂O₂ after inhibition by PH₃. Insect organelles released threefold more H₂O₂ than did mouse organelles, when exposed to PH₃. Production of H₂O₂ by PH₃-treated insect mitochondria was increased significantly on addition of the substrate α-glycerophosphate. Succinate did not enhance H₂O₂ production, however, indicating that the H₂O₂ did not result from the autoxidation of ubiquinone. NAD+-linked substrates, malate and pyruvate also had no effect on H₂O₂ production, suggesting that NADH-dehydrogenase was not the source of H₂O₂. Data obtained using antimycin and myxothiazol, both of which stimulated the release of H₂O₂ from insect mitochondria, lead to the conclusion that glycerophosphate dehydrogenase is a source of H₂O₂. The effect of combining PH₃, antimycin, and myxothiazol on cytochrome spectra in insect mitochondria was also recorded. It was observed that PH₃ reduces cytochrome c oxidase but none of the other cytochromes in the electron transport chain. There was no movement of electrons to cytochrome b when insect mitochondria are inhibited with PH₃. The spectral data show that the inhibitors interact with the respiratory chain in a way that would allow the production of H₂O₂ from the sites proposed previously.