The murine melanoma cell line B16. F10 (H-2 b) was used to study specific T cell responses that reject tumors. Stable B16 transfectants were established that express viral Ags, either the hepatitis B surface Ag (HBsAg) or the large tumor Ag (T-Ag) of SV40. B16 cells and their transfected sublines were CD40+ CD44+ but expressed no (or low levels of the) costimulator molecules CD154 (CD40L), CD48, CD54, CD80, and CD86. Surface expression of MHC class I (K b, D b) and class II (IA b) molecules by B16 cells was low, but strikingly up-regulated by IFN-γ. CD95 (Fas) and CD95 ligand (CD95L (FasL)) were “spontaneously” expressed by B16 cells growing in vitro in serum-free medium; these markers were strikingly up-regulated by IFN-γ. B16 cells coexpressing CD95 and CD95L were irreversibly programed for apoptosis. In vitro, noninduced B16 transfectants stimulated a specific IFN-γ release response, but no cytolytic response (in a 4-h assay) in MHC class I-restricted CTL; in contrast, IFN-γ-induced B16 targets were efficiently and specifically lysed by CTL. In vivo, B16 transfectants were specifically rejected by DNA-vaccinated syngeneic hosts through a T-dependent immune effector mechanism. The tumors showed evidence of massive apoptosis in vivo during the rejection process. The data suggest that CTL-derived IFN-γ enhances an intrinsic suicide mechanism of these tumor cells in addition to facilitating lytic interactions of effectors with tumor targets.