Alloxan can act as a generator of reactive oxygen species (ROS) as long as sufficient suitable reducing agents (e.g. reduced glutathione) and oxygen are available. Using electron spin resonance-spectroscopy and the oxygen-centered spin trap DEPMPO, we demonstrate that hydroxyl radicals (OHzrad;) are formed in vitro by alloxan in the presence of glutathione (GSH) and chelated divalent iron. Furthermore, peroxidation of polyunsaturated fatty acids from phosphatidylcholine-containing liposomes with concomitant formation of malondialdehyde (MDA) was used as a further indicator for a preceding OHzrad; formation. Melatonin, the main secretory product of the pineal gland, is an effective scavenger of OHzrad;. The 50%-inhibitor concentration (IC50-value) for melatonin to scavenge OHzrad; generated from the alloxan/GSH-reaction in the presence of ferrous ions was 23 μmol/L. In contrast to the ability to effectively scavenge OHzrad;, the potential of melatonin to prevent lipid peroxidation is considerably less pronounced.