The application of AAV2 or AAV8 vectors for delivery of human coagulation factor IX (hF.IX) is a promising gene therapy for hemophilia B. One major limitation of this therapy is the development of antibodies and a cytotoxic T lymphocyte (CTL) response against both the vector capsid and the transgene. We determined the class I and class II MHC peptide epitopes for AAV2, AAV8, and hF.IX after administration of AAV-2-hF.IX or AAV8-hF.IX in H2b (C57BL/6), H2d (BALB/c), or H2k (C3H) strains of mice. The results indicate that the AAV2 capsid peptide AA373-381, the AAV8 capsid peptide AA50-58, and the hF.IX transgene peptide AA311-319 can elicit a CTL response as indicated by an IFN-γ ELISPOT assay and an in vivo CTL assay. Furthermore, a strong H2k MHC II-restricted Th1 response can be elicited in C3H mice by the AAV8 capsid peptide AA126-140 and the hF.IX peptide AA108-122, whereas a strong Th2 response can be elicited by the AAV2 peptide AA475-489. These results show that specific CTL responses are generated to both AAV capsid epitopes and hF.IX epitopes after injection of AAV-hF.IX, and MHC class II epitopes derived from AAV-hF.IX promote development of either Th1 or Th2 cells. Copyright © The American Society of the Gene Therapy.