Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8⁺ CTLs. MHC-restricted CD8⁺ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8⁺ CTL responses are modulated by CD4⁺ helper T cells and by antigen-presenting cells. In this study, highly purified CD8⁺ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8⁺ T cells. Partial MHC class I matching of tumor and CD8⁺ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8⁺ CTLs. A1- and A2-restricted CD8⁺ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8⁺ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.