The lytic activity of CD8+ cytotoxic T lymphocyte (CTL) cell lines or clones can be inhibited by addition of the peptide recognized by these cells. The mechanisms underlying this phenomenon are not fully understood. Here we have analyzed peptide-induced CTL paralysis using in vivo generated ovalbumin (OVA)-specific CTL. Lytic activity of OVA-specific CTL was inhibited by addition of the immunodominant OVA-peptide SIINFEKL in a dose-dependent manner. Paralysis was induced rapidly and binding of the peptide to MHC class I molecules was required. Using mixing experiments with CTL populations of different peptide specificities restricted to the same MHC class I molecule we identified a veto-like mechanism: the cytotoxic activity of the peptide-recognizing CTL was inhibited while the lytic activity of the peptide-presenting CTL was unaltered. Only CD8+ CTL but not CD4+ T cells or B+ cells induced paralysis. After removal of the peptide-presenting CTL by magnetic cell sorting, paralysis was maintained and paralyzed CTL showed no signs of apoptosis. Loss of cytotoxicity could be induced in CTL populations from Fas-deficient (lpr+/lpr+) or Fas ligand-deficient (gld+/gld+) mice and mixtures thereof, implying that Fas/Fas ligand interactions are not involved during induction of paralysis. Hence, peptide-induced paralysis of CTL is due to a self-veto mechanism rather than to mutual killing of CTL. These findings may have implications for in vivo immunization with peptides, viral escape and peripheral tolerance mechanisms.