Phosphine (PH(3)), a widely used pesticide, was found in our recent study to induce oxidative damage in the brain, liver and lung of rats. We also observed that melatonin significantly blocked this action. The present study focused on brain and the magnitude and mechanism of protection of PH(3)-induced oxidative damage by melatonin in vitro and in vivo. PH(3) in whole brain homogenate (3 mg protein/mL Tris-HCl pH 7.4 buffer) induced increasing lipid peroxidation [as malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA)] dependent on concentration (0.25-2 mM) and time (30-150 min), reaching a maximum level of 2.9-fold at 90 min after PH(3) at 1 mM. Elevation of MDA + 4-HDA levels by PH(3) at 1 mM was also observed in homogenates of cerebral cortex, cerebellum, hippocampus and hypothalamus examined individually. Melatonin at 0.1-2 mM progressively inhibited PH(3)-induced lipid peroxidation in brain and regions thereof. Additionally, PH(3) induced brain DNA oxidation in vitro and in vivo determined as 8-hydroxyguanosine (8-OH-dG). Melatonin at 1 mM significantly suppressed PH(3)-induced brain DNA oxidation in vitro. PH(3) at 4 mg/kg i.p. significantly elevated 8-OH-dG in frontal cortex and melatonin prevented it. PH(3) in vivo marginally lowered brain glutathione peroxidase activity and melatonin restored it completely. In contrast, PH(3) and melatonin both stimulated superoxide dismutase production. Brain glutathione (GSH) levels in PH(3)-treated rats were significantly reduced at 30 min and recovered gradually. It is concluded that melatonin, probably because of its free radical scavenging ability, confers marked protection against PH(3)-induced oxidative toxicity in brain.