Human adenoviruses have been developed as an attractive vehicle for in vivo liver-directed gene therapy. Problems with the application of first generation recombinant adenoviruses to liver-directed gene therapy have been transient expression of the recombinant gene and development of hepatitis. Previous studies in mouse models of gene transfer to liver and lung suggested that MHC class I-restricted cytotoxic T lymphocytes (CTLs) to viral antigens may be effectors in the elimination of transgene expression. The goal of this study was to evaluate the importance of viral antigens versus transgene product in inducing CTL mediated hepatocyte destruction in vivo. Immunization of C57BL/6 mice with a lacZ-expressing adenovirus elicited CTL responses to both viral antigens and the transgene product, beta-galactosidase (beta-gal). Adoptive transfer experiments, as well as studies involving lacZ-transgenic mice (ROSA-26) revealed that CTLs to viral antigens are sufficient to destroy virus-infected hepatocytes, indicating that CTLs to beta-gal can not solely account for the observed hepatocyte destruction that has characterized the use of first generation viruses. In addition, we confirmed that B cell-mediated events do not participate in destruction of hepatocytes in vivo, despite the production of virus- and beta-gal-specific antibodies. These data confirm the hypothesis that viral gene expression elicits host responses that contribute to the problem of transgene instability. Recombinant adenoviruses must be redesigned to diminish viral gene expression if they are to be used in the treatment of chronic diseases.