We have previously identified mutated ras peptides reflecting the glycine to valine substitution at position 12 as HLA-A2-restricted, CD8+ CTL neo-epitopes. CTL lines produced against these peptide epitopes lysed the HLA-A2+ Ag-bearing SW480 primary colon adenocarcinoma cell line, although IFN-γ treatment of the targets was necessary to achieve efficient cytotoxicity. Here, we compared the lytic phenotype of the SW480 cell line to its metastatic derivative, SW620, as an in vitro paradigm to further characterize the nature of a HLA class I-restricted, Ag-specific CTL response against neoplastic cell lines of primary and metastatic origin. Although both colon carcinoma cell lines were lysed by these Ag-specific CTL following IFN-γ pretreatment, the mechanisms of lysis were distinct, which reflected differential levels of sensitivity to the Fas pathway. Whereas IFN-γ pretreatment rendered SW480 cells sensitive to both Fas-dependent and-independent (perforin) pathways, SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. Moreover, pretreatment of SW480 cells with the anti-colon cancer agent, 5-fluorouracil (5-FU), led to enhanced Fas and ICAM-1 expression and triggered Ag-specific CTL-mediated lysis via Fas-and perforin-based pathways. In contrast, these phenotypic and functional responses were not observed with SW620 cells. Overall, these data suggested that 1) IFN-γ and 5-FU may enhance the lytic sensitivity of responsive colon carcinoma cells to immune effector mechanisms, including Fas-induced lysis; 2) the malignant phenotype may associate with resistance to Fas-mediated lysis in response to Ag-specific T cell attack; and 3) if Ag-specific CTL possess diverse lytic capabilities, this may overcome, to some extent, the potential “escape” of Fas-resistant carcinoma cells.