Peroxynitrite, a biological oxidant, can induce lipid peroxidation in biological membranes which leads to the formation of various hydroperoxides. Here, we report the formation of singlet oxygen (1O2) in the reaction of peroxynitrite with tert-butyl hydroperoxide (t-BOOH) used as a model compound for organic hydroperoxides. The formation of singlet oxygen was observed by (i) dimol emission in the red spectral region, (ii) monomol emission in the infrared region at 1270 nm and by (iii) chemical trapping of singlet oxygen with anthracene-9,10-diyldiethyl disulfate (EAS). The emission signal was increased when H2O was replaced by deuterium oxide and was quenched by sodium azide. When singlet oxygen was generated in the reaction of peroxynitrite with t-BOOH, the 1O2 quenching rate constant for sodium azide was estimated from a Stern-Volmer plot as 1.3 x 10(8) M(-1) S(-1) which is in line with known values. The 1O2 generation in the peroxynitrite/t-BOOH reaction was also detected by the formation of the endoperoxide of EAS. These data establish the generation of 1O2 in the reaction of peroxynitrite with t-BOOH and suggest a potential involvement of 1O2 in peroxynitrite-mediated reactions in biological systems.