In order to better understand the broad applicability of adenovirus (Ad) as a vector for human vaccine studies, we compared four adenovirus (Ad) vectors from families C (human serotype 5, AdHu5), D (human serotype 26, AdHu26) and E (chimpanzee serotypes 6 and 7, AdC6 and AdC7) of adenovirideae. Seroprevalence rates and neutralizing antibody titers to the two human origin Ad viruses were found to be higher than previously reported, especially in countries of Sub Saharan Africa. Conversely, prevalence rates and titers to AdC6 and AdC7 viruses were markedly lower. Healthy human adults from the US had readily detectable circulating T cells to Ad viruses, which in some individuals were unexpectedly high in response to AdHu26 virus. The magnitude of T cell responses to the AdHu5 virus correlated with those to AdHu26 virus, suggesting T cell recognition of conserved epitopes. In mice all of the different Ad vectors induced CD8(+) T cell responses that were comparable in magnitude and cytokine production profile. Prime-boost regimens comparing different combinations of Ad vectors failed to indicate that the sequential use of Ad vectors from distinct families resulted in higher immune responses than the use of serologically distinct Ad vectors from the same family. Moreover, the transgene product-specific antibody responses induced by AdHu26 and AdC vectors were markedly lower than those to an AdHu5 vector. AdHu26 vectors, and to a lesser extent AdC vectors, induced more potent Ad neutralizing antibody responses. These results suggest that the potential of AdHu26 as a vaccine vector may suffer similar limitations as those found for vectors based on other prevalent human Ad viruses.