In the Jurkat T cell line, triggering of the TCR leads to activation of phospholipase C, resulting in an increase in inositol trisphosphate (IP3) release followed by a rise in intracellular Ca2+. This signaling pathway is interrupted by cholera toxin (CTX) treatment. To possibly explain this inhibition, we demonstrate that CTX can affect the TCR/CD3 complex itself by causing a covalent modification of the CD3-zeta subunit. After exposure of Jurkat cells to CTX, CD3-zeta increases its apparent m.w. and becomes more acidic in isoelectric focusing. The time course of the modification correlates well with the reduction in IP3 generation and Ca2+ release after CTX treatment, suggesting that the modification of zeta might be the cause of the impaired TCR/CD3 signaling. As is true for the CTX-mediated decrease in TCR signaling, the change in CD3-zeta was cAMP-independent and cannot be evoked by the enzymatically inactive CTX-B subunit alone.