Replication-defective adenovirus (ADV) and poxvirus vectors have shown potential as vaccines for pathogens such as Ebola or human immunodeficiency virus in nonhuman primates, but prior immunity to the viral vector in humans may limit their clinical efficacy. To overcome this limitation, the effect of prior viral exposure on immune responses to Ebola virus glycoprotein (GP), shown previously to protect against lethal hemorrhagic fever in animals, was studied. Prior exposure to ADV substantially reduced the cellular and humoral immune responses to GP expressed by ADV, while exposure to vaccinia inhibited vaccine-induced cellular but not humoral responses to GP expressed by vaccinia. This inhibition was largely overcome by priming with a DNA expression vector before boosting with the viral vector. Though heterologous viral vectors for priming and boosting can also overcome this effect, the paucity of such clinical viral vectors may limit their use. In summary, it is possible to counteract prior viral immunity by priming with a nonviral, DNA vaccine.