The pineal hormone melatonin has neuroprotective effects in a large number of models of neurodegeneration. Melatonin crosses the blood–brain barrier, shows a decrease in its nocturnal peaks in blood with age that has been associated with the development of neurodegenerative disorders, and has been shown to be harmless at high concentrations. These properties make melatonin a potential therapeutic agent against neurodegenerative disorders but the pathways involved in such neuroprotective effects remain unknown. In the present report we study the intracellular pathways implicated in the complete neuroprotection provided by melatonin against glutamate-induced oxytosis in the HT22 mouse hippocampal cell line. Our results strongly suggest that melatonin prevents oxytosis through a direct antioxidant effect specifically targeted at the mitochondria. Firstly, none of the described transducers of melatonin signalling seems to be implicated in the neuroprotection provided by this indole. Secondly, melatonin does not prevent cytosolic GSH depletion-dependent increase in reactive oxygen species (ROS), but it totally prevents mitochondrial ROS production despite the fact that the latter is much higher than the former. And finally, there is a high correlation between the concentration at which melatonin and closely related indoles exert a direct antioxidant effect in vitro and a neuroprotective effect against glutamate-induced oxytosis.