The absence of preexisting immunity against porcine adenovirus (Ad) serotype 3 (PAd3) and bovine Ad serotype 3 (BAd3) in humans makes them attractive alternatives to human Ad serotype 5 (HAd5) vectors. To determine whether there is significant cross-reactivity among HAd5, BAd3 and PAd3 at the level of cell-mediated immune responses, BALB/c mice were inoculated intraperitoneally with wild-type (WT) or replication-defective (RD) HAd5, BAd3 or PAd3. After 35 days of the first inoculation, cross-reactive CD8+ cytotoxic T cells, as well as CD4+ Th1- and Th2-helper T cells, in the spleen were analyzed by enzyme-linked-immunospot, flow cytometry and cytotoxic T lymphocyte assays. Virus-neutralization assays were used to evaluate humoral cross-reactivity. CD8+ or CD4+ T cells primed with WT or RD HAd5, PAd3 or BAd3 showed significant (P<0.005) reactivity with homologous Ad antigens, whereas only minimal cross-reactivity was observed on stimulation with heterologous Ad antigens. Ad-neutralizing antibodies were found to be homologous Ad specific. Overall, these results suggest that there is no significant immunological cross-reactivity among HAd5, BAd3 and PAd3, thereby supporting the rationale for the use of BAd3 and PAd3 as alternative HAd vectors to circumvent anti-HAd immunity in humans.