Although pre-clinical results using recombinant adeno-associated virus (rAAV) vectors as vaccine carriers for HIV-1 have been encouraging, clinical trials for rAAV vectors encoding HIV-1 antigens have yielded disappointing results. We therefore tested rAAV vectors of different serotypes expressing gag of HIV-1 for induction of transgene product-specific CD8+ T cells in mice. The vectors stimulate gag-specific CD8+ T cells, but these cells are unable to expand after a booster immunization with a replication-defective adenoviral (Ad) vector expressing gag. The immuno-inhibitory effect of rAAV priming observed with different AAV serotypes is transgene product-specific, independent of the interval between prime and boost, and extends to boosts with vaccine modalities other than Ad vectors. rAAV vector-induced CD8+ T cells proliferate poorly, produce low levels of IFN-gamma in response to gag, have upregulated immuno-inhibitory molecules, and do not protect efficiently against challenge with a surrogate pathogen. The impairment of the T cells' proliferative capacity is caused by persistence of the antigen-encoding rAAV vectors that is reversed by transfer of CD8+ T cells into an antigen-free environment. These data suggest that rAAV vectors induce functionally exhausted T cells and could dampen the immune response to a natural infection.