Bilirubin is a bile pigment that may have an important role as an antioxidant. Its antioxidant potential is attributed mainly to the scavenging of peroxyl radicals. We investigated the reaction of bilirubin with peroxynitrite in phosphate buffer and in blood plasma. In phosphate buffer bilirubin was rapidly oxidized by micromolar concentrations of peroxynitrite, and its oxidation yield was higher at alkaline pH with an apparent pKa = 6.9. In contrast, the major oxidation product of bilirubin in plasma was biliverdin, and the pH profile of its oxidation yield showed a slightly increased oxidation at acidic pH without a clear inflection point. The addition of NaHCO3 to bilirubin decreased the peroxynitrite-dependent oxidation, suggesting that the reactive intermediates formed in the reaction between CO2 and peroxynitrite are less efficient oxidants of bilirubin. The antioxidant role of bilirubin was investigated in some peroxynitrite-mediated plasma protein modifications that are enhanced by CO2 (tryptophan oxidation and protein tyrosine nitration) or slightly decreased by CO2 (protein carbonyl groups). Bilirubin in the micromolar concentration range afforded a significant protection against all these oxidative modifications and, notably, protected plasma proteins even when the pigment was added 5 s after peroxynitrite (i.e., when peroxynitrite is completely decomposed). The loss of tryptophan fluorescence triggered by peroxynitrite was a relatively slow process fulfilled only after a few minutes. After this time, bilirubin was unable to reduce the tryptophan loss, and it was unable to reduce previously formed nitrated albumin or previously formed carbonyls. We deduce that bilirubin in plasma cannot react to a significant extent with peroxynitrite, and we suggest that bilirubin, through a hydrogen donation mechanism, participates as a scavenger of secondary oxidants formed in the oxidative process.