This study assessed the mucosal and systemic immune responses following repetitive adenoviral vector instillation to the parotid glands. Also, we investigated the feasibility of oral tolerance induction as a rational strategy to overcome the immunological reactions. The replication-deficient recombinant adenovirus vector AdCMVCAT was instilled into rat parotid glands. Chloramphenicol acetyltransferase (CAT) activity in the parotid was observed after a first or second AdCMVCAT infection, but not after a third vector administration. ELISA assays showed increased anti-adenovirus immunoglobulin G (IgG) and IgM in serum, and also anti-adenovirus IgA in gland extracts and saliva after virus administration. The results of in vivo neutralization experiments demonstrated that salivary IgA and IgM prevented reinfection of the parotids with adenoviral vectors. Subsequently, studies were conducted to induce tolerance to adenovirus by peroral feedings of ultraviolet (UV)-inactivated virus before gene administration to the parotid glands. Between 3 and 13 doses of virus were fed to rats. Final parotid gene expression was dependent on the number of viral feedings and the amount fed. Tolerized animals showed prolonged and heightened gene expression in the salivary glands compared to control animals and displayed gene expression even after three administrations of vector. Mononuclear cells from the spleens of these animals showed reduced proliferation following adenovirus stimulation. This same cell population was depleted of CD8+ T cells and found to produce less interferon-γ (IFN-γ) after virus challenge. This profile indicates the down regulation of Th1 cell-mediated responses. These results indicate that oral tolerance induction is a potentially useful adjunct to virus-based gene therapy.