Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8+ T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8+ T cells increased ROI levels in a manner dependent upon Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5, 5-dimethyl-1, 3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, and proliferation of naive CD8+ and CD4+ T cells. We also found that TNF-α production by effector and memory CD8+ T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-γ. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8+ T cells are able to modulate signaling, proliferation, and function.