To prevent uncontrolled expansion, the massive proliferation of T cells during an acute immune response has to be followed by controlled deletion. Here we show that similar to Fas, perforin is not only an important effector molecule of cytotoxic T lymphocytes (CTL) but also involved in down-regulating peripheral T cells. Mice deficient for both the CTL effector molecule perforin and the apoptosis-inducing Fas ligand spontaneously develop infiltration of highly activated CD8(+) T cells in kidney and liver and die between 5 and 12 weeks of age. Injection of staphylococcal enterotoxin B (SEB) into perforin-deficient mice results in dramatically increased selective expansion and prolonged persistence of CD8(+), but not CD4(+), SEB-reactive T cells. Also, secondary immunization of TCR transgenic perforin-deficient mice with the lymphocytic choriomeningitis virus glycoprotein-derived epitope peptide leads to an increased proliferation of transgenic CD8(+) T cells, that is not explained by failure to deplete professional antigen-presenting cells. These results point to a novel mechanism of T cell homeostasis in which the acquisition of perforin-dependent cytotoxic activity regulates the expansion and persistence of CD8(+) effector T cells in vivo.