The pancreas is an ideal organ for adenoviral gene therapy because of the high level of gene transfer that can be achieved and because of the many diseases that can potentially be treated using this technology. In this report, we characterize the immune response to direct pancreatic injection of adenovirus and we overcome some of the limitations it imposes by using immunosuppression. Direct injection of recombinant adenovirus into the pancreas leads to the production of neutralizing antibodies and to sensitized splenocytes which engage in increased cytotoxic, lymphoproliferative, and cytokine release activity when reexposed to adenovirus. Transgene expression is transient and the vector cannot be readministered. Deletion of CD4+ T helper cells improves expression over time (40% of pancreatic cells express transgene at day 28 vs. 5% in controls), and allows the vector to be readministered in the pancreas, albeit, inefficiently, when compared to naive animals. Similarly, blockade of CD40 ligand, which preserves the CD4+ T helper cell population, also improves expression over time (30% of pancreatic cells express transgene at day 28), and allows the vector to be readministered. With both approaches, neutralizing antibodies are decreased and the remaining splenocytes do not engage in activated immune responses. Thus, local delivery of the adenoviral vector induces a systemic response that prevents pancreatic readministration, even with direct injection. Blockade of CD40 ligand and T helper cell depletion are transient regimens that induce systemic immunosuppression. Until the development of newer strategies that selectively suppress adenoviral immune responses, these are viable alternatives for enhancement of pancreatic adenoviral delivery.