Barbituric acid (2,4,6-pyrimidinetrione) can be transformed by a non-enzymatic hydroxylation into alloxan (2,4,5,6-pyrimidinetetrone). This transformation can be used as a reaction indicating the formation of hydroxyl radicals (.OH). This conversion was detected using HPLC. Formation of .OH was demonstrated by electron spin resonance (ESR) spectroscopy combined with spin-trapping techniques. It was shown that .OH generated via the Fenton reaction abstracts first a hydrogen atom from barbituric acid (BA) and forms intermediately a paramagnetic derivative of BA. After a second attack by another .OH, the BA radical is transformed into dialuric acid (DA), which autoxidizes via the alloxan radical (.ALX) to ALX. Superoxide radicals (.O2-) are formed during autoxidation of DA and.ALX. They are able to regenerate ferrous ions. As a result, traces of iron salts are capable of catalyzing the conversion of large amounts of BA into ALX. Several scavengers of .OH were tested with regard to their efficiency in preventing the transformation of BA into ALX. Of all the scavengers analyzed, melatonin was shown to be one of the most potent compounds.