Replication-deficient adenoviruses (recombinant adenovirus (rec-AdV)) expressing different transgenes are widely used vectors for gene therapy and vaccination. In this study, we describe the tolerization of transgene-specific CTL following administration of β-galactosidase (βgal)-recombinant adenovirus (Ad-LacZ). Using MHC class I tetramers to track βgal-specific CTL, we found that a significant expansion of βgal-specific CTL was restricted to a very narrow dose range. Functional analysis revealed that adenovirus-induced βgal-specific CTL produced only very low amounts of effector cytokines and were unable to exhibit cytolytic activity in a 51 Cr release assay. Furthermore, Ad-LacZ vaccination failed to efficiently clear established βgal-positive tumors. The impaired function of Ad-LacZ-induced CTL correlated with the presence of persisting βgal Ag in the liver. A further increase in the peripheral Ag load by injection of Ad-LacZ into SM-LacZ transgenic mice which express βgal as self-Ag exclusively in peripheral nonlymphoid organs, resulted in the physical deletion of βgal-specific CTL. Our results indicate first that CTL deletion in the course of adenoviral vaccination is preceded by their functional impairment and second, that the outcome of rec-AdV vaccination depends critically on the Ag load in peripheral tissues.