Autoimmune-prone MRL/lpr mice, homozygous for the lpr mutation, exhibit defective apoptosis and develop generalized lymphoproliferation with the accumulation of a double-negative (DN: CD4- CD8-) T cell population. The capacity of lpr T lymphocytes to effectuate Fas- and perforin-mediated cytotoxicity was investigated. Spleen and lymph nodes cells spontaneously lyse Fas- targets (thymocytes) through a Fas-mediated mechanism as a consequence of their overexpression of Fas ligand (FasL) confirmed by semiquantitative reverse transcription (RT)-PCR and immunoprecipitation analysis. This cytotoxicity was greatly increased after stimulation of the effectors by phorbol myristate acetate (PMA) + ionomycin. Under these conditions, MRL/lpr spleen and LN cells exhibited strong Fas-mediated Ca2+-independent cytotoxic activity against wild-type Fas+ (H-2 compatible or incompatible) thymocytes or lipopolysaccharide (LPS)-transformed blast cells. Such Fas-mediated cytotoxic activity was also observed with C57BL/6-lpr, but never with wild-type C57BL/6 or MLR+/+ effectors. Depletion experiments showed that the effector cells of this Fas-mediated cytotoxicity were DN T cells. This subset, which represent in vivo activated T cells, can spontaneously lyse Fas+ targets by a mechanism that does not need the interaction of the T cell receptor (TCR) with major histocompatibility complex molecule plus antigen. This lytic potential is increased by PMA + ionomycin, which sends a second activation signal to these primed T cells. Therefore, the small amounts of Fas receptor expressed on MRL/lpr tissues may account for their nonspecific autoimmune attack by DN cells. In Con A-containing medium, which allows detection of the perforin-mediated pathway against Fas targets, cytotoxic CD8+ effectors were detected that are able to kill lpr thymocytes via a Ca2+-dependent pathway. Thus, in MRL/lpr mice, these CD8+ cells could constitute potent cytotoxic effectors against cells presenting antigen to their TCR.