Peroxynitrite (ONOO-), a biologically active species, can induce lipid peroxidation in biological membranes, thereby leading to the formation of various hydroperoxides. We report herein on the formation of singlet molecular oxygen [O-2 ((1)Delta(g))] in the reaction of peroxynitrite with linoleic acid hydroperoxide (LAOOH) or O-18-labeled LAOOH. The formation of O-2 ((1)Delta(g)) was characterized by (i) dimol light emission in the red spectral region (lambda > 570 nm) using a red-sensitive photomultiplier; (ii) monomol light emission in the near-infrared region (lambda = 1270 nm) with a liquid nitrogen-cooled germanium diode or a photomultiplier coupled to a monochromator; (iii) the enhacing effect of deuterium oxide on chemiluminescence intensity, as well as the quenching effect of sodium azide; and (iv) chemical trapping of O-2 ((1)Delta(g)) or O-18-labeled O-2 ((1)Delta(g)) with the 9,10-diphenylanthracene (DPA) and detection of the corresponding DPAO(2) or O-18-labeled DPA endoperoxide by HPLC coupled to tandem mass spectrometry. Moreover, the presence of O-2 ((1)Delta(g)) was unequivocally demonstrated by a direct spectral characterization of the near-infrared light emission attributed to the transition of O-2 ((1)Delta(g)) to the triplet ground state. For the sake of comparison, O-2 ((1)Delta(g)) deriving from the thermolysis of the endoperoxide of 1,4-dimethylnaphthalene or from the H2O2/hypochlorite and H2O2/ molybdate systems were also monitored. These novel observations identified the generation of O-2 ((1)Delta(g)) in the reaction of LAOOH with peroxynitrite, suggesting a potential O-2 ((1)Delta(g))-dependent mechanism that contributes to cytotoxicity mediated by lipid hydroperoxides and peroxynitrite reactions in biological systems.