Exposure of human T-lymphocytes to heat downregulates TCR zeta chain expression and inhibits (TCR)/CD3-mediated production of inositol triphosphate and [Ca(2+)](i) signaling. Here we investigated whether oxidative stress is involved in the heat-induced downregulation of TCR/CD3-mediated signaling. To this end, we have studied the effect of a thiol antioxidant, N-acetyl-L-cysteine (NAC), and a non-thiol antioxidant, allopurinol, on heat-induced downregulation of TCR/CD3-mediated signaling. We found that preincubation of cells with 10mM NAC significantly reversed the downregulation of TCR/CD3-mediated [Ca(2+)](i) response and restored the suppression of TCR zeta chain protein expression as well as prevented its increased membrane distribution in heat-treated cells. NAC also reversed the downregulation of TCR zeta chain mRNA expression and the active 94kDa TCR zeta chain transcription factor, Elf-1, in heat-treated cells. Consistent with the increase in the TCR zeta chain, preincubation with NAC increased the levels of antigen receptor-induced tyrosine phosphorylation of several cytosolic proteins. Finally, treatment with NAC was able to reverse the suppression of IL-2 production in heat-treated cells. Inactive analog, N-acetylserine, failed to reverse the heat-induced downregulation of TCR/CD3-mediated signaling. Allopurinol, another potent non-thiol antioxidant, also restored the TCR/CD3-mediated [Ca(2+)](i) response in heat-treated cells. These results demonstrate that antioxidants restore the expression of TCR zeta chain and reverse the TCR/CD3-mediated signaling abnormalities associated with heat stress and suggest that heat shock-induced oxidative stress is a mediator of the heat-induced biochemical damage that leads to downregulation of signaling in human T-lymphocytes.