BACKGROUND: We recently reported the isolation and sequencing of 30 novel adenoviruses from chimpanzees, bonobos and gorillas. These adenoviruses are promising candidates for the purpose of expanding the repertoire of adenoviral serotypes that can be used to create vectors for circumventing pre-existing neutralizing antibodies in human populations. We thus aimed to create vectors from 20 of the newly isolated adenoviruses. METHODS: Plasmid molecular clones were created that harbored the complete E1-deleted genomes from 20 of the newly isolated ape adenoviruses belonging to species B, C and E. The plasmids were transfected into human embryonic kidney (HEK) 293 cells to rescue vectors. We also tested normal human sera to determine the extent of pre-existing cross-neutralizing anti-adenovirus neutralizing antibodies. RESULTS: Twelve vectors could be rescued and expanded following transfection into HEK 293 cells with yields (from fifty 150-mm culture dishes) that ranged from 3 × 10(11) to 7 × 10(13) viral particles. Sera from 50 normal human donors were tested for the presence of neutralizing activity against 21 of the newly isolated ape adenoviruses. Cross-neutralizing activity was generally low, although outliers with high neutralizing activity were frequently detected. Species B ape adenoviruses generally showed the least cross-neutralization with antibodies present in the human sera that were tested. CONCLUSIONS: E1-deleted adenovirus vectors can be created from a wide variety of ape adenoviruses that can be rescued and propagated in HEK 293 cells. The prevalence of pre-existing antibodies that can neutralize these adenoviruses in human populations is low.