We have investigated the role of cytokines (IL-2, IL-3, IL-4, IL-6, IFN-gamma, and GM-CSF) in the generation of primary cytotoxic T lymphocytes (CTL), within a single tumor system. The murine carcinoma line 1 was transfected with expression vectors with cDNA for these cytokines. Line 1 expresses low levels of class I MHC molecules, but can be induced with dimethyl sulfoxide or IFN-gamma to express high levels of class I. Class I low line 1 cells are not susceptible to CTL lysis, while class I high line 1 are lysed by CTL, which allows us to assay for CTL activity. To isolate primary CTL, tumor-infiltrating lymphocytes were isolated from cytokine-expressing or control tumors, growing in vivo. Most cytokines stimulated nonspecific killers, but IL-2 and IL-3 stimulated primary CTL. While IFN-gamma alone did not generate primary CTL, coexpression of IFN-gamma with IL-2 resulted in CTL generation. This is the first comparison of the effects of a series of cytokines on primary CTL development, and has important implications for vaccine development and immunotherapy.