We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor alpha [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunothera 38:281]. In this report we demonstrated that ProT alpha, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumor-specific CD8+ cytotoxic T lymphocytes (CTL). The ProT alpha-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only ProT alpha developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving ProT alpha and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The ProT alpha-induced CD8+ CTL activity was regulated by ProT alpha-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8(+)-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both ProT alpha and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that ProT alpha is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since ProT alpha may prove to be useful in clinical protocols aimed at cancer immunotherapy.