Peroxynitrite (ONOO−) is an endogenous molecule, formed by rapid coupling between NO and O2−. ONOO− is known to be a strong oxidant of thiols and metalloorganic compounds and also a nitrating agent of aromatic compounds such as tyrosine. However, its chemistry is not yet well elucidated under physiological conditions. Melatonin, which is an indole-amine produced by the pineal gland and other organs, has antioxidant properties. We show that melatonin reacts with ONOO− in phosphate-buffered solutions. We provide evidence of nitrosation and oxidation at the pyrrole nitrogen leading to 1-nitrosomelatonin and 1-hydroxymelatonin, these being the major reactions in aqueous phosphate-buffered solutions besides other aromatic hydroxylations and nitration. 4-Nitromelatonin is formed, but in small amounts. The kinetics of all transformations were strictly dependent on ONOO− decay, whereas yields varied with pH and the presence of CO2. The N-oxidation became competitive with nitrosation at pH 7.4, in medium containing a sufficient amount of CO 2. A proposed mechanism involves the transient formation of melatonyl radical and ONOO radical derived from ONOO− decay.