Despite the low and short expression of secondary Ag, prime-boost immunizations using homologous or heterologous vectors are capable of amplifying memory CD8+ T cells. This is mainly attributed to the rapid presentation of Ag by APCs and the high proliferative capacity of memory CD8+ T cells. Nevertheless, certain viruses and vectors often require prolonged Ag presentation for optimal T cell priming, and the influence of such a prolonged presentation during secondary immune induction is not clear. To address this issue, we primed and boosted mice intradermally (i.d.) with plasmid DNA that was recently reported to require prolonged Ag presentation for maximal CD8+ T cell priming. Although functional memory CD8+ T cells were present in the mice after i.d. priming, the secondary CD8+ T cell response elicited was limited and reached a similar level of that observed during priming. The initial levels of secondary Ag expressed in the boosted mice were sufficient to prime CD8+ T cell response in naive hosts, suggesting that lower Ag load alone does not explain the limited secondary immune responses observed. Removal of the injection site 5 or 10 days after i.d. boosting immunization resulted in diminished Ag presentation and no expansion of memory CD8+ T cells. In fact, Ag-presenting activity following boost occurred mainly two weeks postimmunization, a time when the Ag was no longer expressed in situ. These findings suggest that when the boosting vector triggers prolonged Ag presentation, the lack of synchronicity between Ag accessibility and Ag presentation limits secondary immune responses.