This study demonstrates that a syngeneic specific cytotoxic T lymphocyte (CTL) response to a class I major histocompatibility complex (MHC) positive tumour requires dual processing and recognition of tumour antigens. One type of antigen is processed and expressed in association with class I MHC at the surface of intact tumour cells. It is recognized by CD8 alpha, beta TCR CTL in vitro and by protective immune T cells in vivo and thus functions as a tumour-associated transplantation antigen (TATA). The other type of antigen is processed and expressed by distinct host APC in association with class II MHC. This is recognized by immune CD4 T cells which function as essential helper cells in the generation of the CD8 CTL response. These conclusions are supported by cell depletion and reconstitution experiments as well as by blocking experiments with monoclonal antibodies using the highly metastatic class II negative murine lymphoma ESb as a model system. The existence of two types of cognate T cell responses in a syngeneic anti-tumour response was directly proved by the establishment of two types of tumour specific T cell lines which required as co-stimulator either MHC class II positive APC or IL-2. In suboptimal mixed lymphocyte tumour cell cultures either of these co-stimulator functions was found to be limiting the overall anti-tumour CTL response. The generation of the tumour specific CTL response could be blocked by monoclonal antibodies against all the molecules involved in the cognate interactions (i.e. class I MHC, CD8, class II MHC, CD4 and TCR) but not by anti-CD2 or anti-IgG. The strict requirement for helper cells and APC could be bypassed by the addition of recombinant IL-2 but optimal triggering of CD8 CTL-precursor required viable tumour stimulator cells. This well characterized in vitro assay may be useful (i) for monitoring the immune status of CD4 and CD8 immune T cells separately, for instance of tumour bearing and/or treated animals and (ii) for the development and testing of potent tumour cell vaccines with T cell stimulatory and/or co-stimulatory activities.