Cytotoxic T lymphocytes are highly resistant to killing by their own cytolytic protein perforin. We have investigated the molecular basis of this self-protection mechanism. CTL withstood high dosages of perforin, when complete lysis was obtained with various tumor target cell lines. A peptide-specific CTL clone readily lysed tumor targets presenting the peptide, but was unable to kill the peptide-presenting CTL in spite of equal degranulation. With streptolysin O, a pore-forming lytic protein of bacterial origin, no difference in susceptibility between the various targets was detected. Perforin was radioactively labeled by using the Bolton and Hunter method without any loss of its lytic activity. Experiments performed at 4 degrees C revealed that, on all of the cells studied, binding of the labeled perforin to the membrane is reversible and strictly Ca(2+)-dependent. After a short exposure to 37 degrees C, perforin was no longer dissociated from cell membranes by EDTA. Although no correlation between susceptibility and the extent of perforin binding was detected, differences in the conformation of membrane-bound perforin were observed. Perforin adsorbed to resistant cells was cleaved by trypsin into a 55-kDa (C-terminal) and a 10- to 15-kDa (N-terminal) fragment, whereas this cleavage was not found on tumor cell-bound perforin. A portion of perforin was included in the Triton X-114 detergent phase at 4 degrees C on resistant CTLs only. Our results are compatible with the notion that a protective molecule, specifically expressed on CTLs, interacts with perforin, thereby rendering it lytically inactive.