Adenoviral vectors can be used to deliver complex Ag to dendritic cells (DC), and thus may be ideal for stimulating broad T cell responses to viral pathogens and tumors. To test this hypothesis in a relevant primate model, we used recombinant adenovirus serotype 5 vectors expressing SIV Gag Ag to transduce monocyte-derived DC from rhesus macaques, and then immunized donor animals either by intradermal or intranodal injections. T cell responses were evaluated by ELISPOT assay using previously frozen PBMC pulsed with pools of 15-mer peptides representing the Gag sequence. Immunization resulted in rapid and potent induction of T cell responses to multiple regions of Gag, with frequencies approaching 1 Gag-specific T cell per 500 uncultured PBMC. Surprisingly, intradermal and intranodal injections generated a similar intensity and breadth of response, indicating that administration of Ag-expressing DC by either route may be equally effective at inducing immune responses. Detailed analysis of two monkeys revealed CD8(+) T cell responses to several peptide epitopes of Gag not previously described, at least two of which are restricted by MHC class I alleles not currently identified. Repeated vaccination did not induce T cell responses to the adenoviral vector and did not prevent Ag-expressing DC injected under the capsule of the lymph node from migrating to the paracortex and interposing between T cells. However, boost injections of adenovirus-transduced DC were generally limited in efficacy. These findings support the use of adenovirus-transduced DC in the therapy of HIV infection and cancer.