Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O₂^•−) with nitric oxide (NO^•) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O₂^•− and NO^• donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO^• donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O₂^•− and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO^•, thymic stromal cells were investigated to determine if they produced NO^•. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1–treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis.