Liver toxicity observed in a clinical trial of adeno-associated virus serotype 2 (AAV2) delivered systemically to patients with hemophilia was ascribed to killing of vector-transduced hepatocytes by capsid-specific T cells. This study evaluated the biology of T cell activation in response to AAV capsids in murine models. CD8⁺ T cell epitopes were mapped to capsids from AAV2, AAV7, and AAV8. A tetramer generated in response to a dominant capsid epitope in BALB/c mice was shared between these AAV serotypes. Administration of AAV2 vector resulted in the activation of capsid-specific CD8⁺ T cells as evidenced by binding to tetramer and production of capsid-induced interferonexpression; this was not observed with the AAV7 and AAV8 vectors. CD8⁺ T cells specific to AAV2 capsids demonstrate functional cytolytic activity in vivo to peptide-loaded target cells. The frequency of capsid-specific T cells was much higher in liver than in blood or spleen. The performance of liver-directed AAV-mediated gene transfer was not diminished in animals with high levels of pre-existing capsid-specific T cells. We conclude that cross-presentation of AAV capsids does result in activation of cytotoxic T lymphocytes (CTLs) in a serotype-specific manner; however, there is no evidence that vector-transduced hepatocytes are targets for CTL effector activity.