We investigated the type of T cell response involved in Meth A tumor rejection in primary immune and hyperimmune syngeneic mice. It was found that a CD4+ T cell-mediated delayed-type hypersensitivity (DTH) response activating non-specific killer cells such as macrophages, NK and LAK cells, without a specific CD8+ cytotoxic T lymphocyte (CTL) response, was the major immune response leading to Meth A tumor rejection in primary immune mice. In contrast, the specific CD8+ CTL response was the major response leading to the tumor rejection, in addition to CD4+ T cell-mediated DTH response, in hyperimmune mice. Analysis of CD4+ T cell clones established from primary immune and hyperimmune spleen cells indicated that a CD4+ T cell clone (C9) of primary immune mice (although only one clone was established) was of Th1 type, and induced cytotoxicity in accessory cells by classic DTH in vitro. Eight CD4+ T cell clones were established from hyperimmune spleen cells. Six out of the eight clones were of the Th2 type and two were Th0-like. However, no Th1-type CD4+ T cell clone was established from hyperimmune spleen cells. All of these CD4+ T cell clones, even the Th2-type clones, were capable of inducing cytotoxicity in vitro in T cell-depleted accessory cells, as in an in vitro DTH response. We postulate on the basis of these results that the T cell response leading to Meth A tumor rejection in vivo sequentially changed from a CD4+ T cell-mediated classic DTH response to a CD8+ CTL response, in addition to a cellular response mediated probably by Th2-type cells, during the process of repeated immunization.