Vaccination with recombinant viral vectors may be impeded by preexisting vector-specific immunity or by vector-specific immunity induced during the priming immunization. It is assumed that virus-neutralizing antibodies represent the principal effector mechanism of vector-specific immunity, while killing of infected cells by vector-specific cytotoxic T lymphocytes (CTLs) has also been suggested. Using recombinant Semliki Forest virus (rSFV) expressing E6E7 antigen from human papillomavirus, we demonstrate that secondary immune responses against E6E7 are neither affected by vector-specific antibodies nor by CTL-mediated killing of infected cells. Instead, the presence of the antigen during the prime immunization appeared to be the main determinant for the boosting efficacy. After priming with rSFVeE6,7, a homologous booster stimulated the primed E6E7-specific CTL response and induced long-lasting memory. Passively transferred SFV-neutralizing antibodies did not inhibit E6E7-specific CTL responses, although transgene expression was strongly reduced under these conditions. Conversely, in mice primed with irrelevant rSFV, induction of E6E7-specific CTLs was inhibited presumably due to vector-specific responses induced by the priming immunization. When during the priming with irrelevant rSFV, E7-protein was co-administered, the inhibitory effect of vector-specific immunity was abolished. These results suggest that, apart from vector-specific antibodies or killing of infected cells, T-cell competition may be involved in determining the efficacy of viral vector-based prime-boost immunization regimens.