This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals. Elimination of CD25-expressing T cells, which constitute 5–10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them. The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4− 8− cytotoxic cells akin to NK cells. Furthermore, in vitro culture of CD25+ 4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4− 8− cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+ 4+ T cells inhibited the generation. In this culture, self-reactive CD25− 4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+ 4+ T cells, secreting large amounts of IL-2. The IL-2 thus produced appeared to be responsible for the generation of CD4− 8− NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4− 8− cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4− 8− cells with CD25− 4+ T cells from IL-2-deficient mice did not. Thus, removal of immunoregulatory CD25+ 4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones. This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans.