The high prevalence of preexisting immunity to the commonly used adenoviral vectors, as well as the requirement for readministration of vector for multiple therapeutic applications, necessitates the development of a panel of immunologically distinct adenoviral vectors against which neutralizing antibodies are rare in human populations. We have completely sequenced three chimpanzee-derived adenoviruses, Pan 5, Pan 6, and Pan 7, and have molecularly cloned E1-deleted vector genomes from each as bacterial plasmids. All the E1-deleted vectors were grown to high titer in HEK 293 cells. Neutralizing antibodies to the chimpanzee adenoviral vectors were not detected in serum samples from human subjects. In vitro cross-neutralization using rabbit antisera and in vivo readministration experiments in mice demonstrated that antibodies against Pan 5, Pan 7, or Pan 9 cross-neutralize one another but do not neutralize Pan 6. These results indicate that chimpanzee adenoviral vectors may be useful as vaccines or gene therapy vectors in human populations and should allow applications that require multiple vector administrations.