Adenoviruses (Ad) have been extensively studied as gene delivery vectors in gene therapy and as vaccine carriers. The cell-mediated cytotoxicity induced by Ad is of great interest in both applications. However, the mechanism underlying Ad-specific cytotoxic T lymphocyte (CTL) generation and effector function remains unclear. In this study, we used a novel MHC class I tetramer and an in vivo CTL assay to examine the role of CD28, perforin, Fas ligand (FasL), and TNF-α in the generation and function of Ad-specific CTLs in vivo. During the primary response, there was a significant defect in both the generation and in vivo effector function of Ad-specific CTLs in CD28-/- mice, but not in CD4+ T cell-depleted mice or CD4-/- mice. The relative role of CTL effector molecules was assayed by in vivo CTL assay in perforin- or FasL-mutant mice, using donor cells from Fas-deficient or TNFR1/TNFR2-deficient mice. The results indicated that the in vivo CTL activity is mediated mainly by perforin. In the absence of perforin, production of FasL, but not TNF-α, by the CTLs results in lower level Ad-specific killing of target cells. These results provide important implications concerning the development of safe and effective Ad vectors for gene therapy and vaccines.