Dendritic cells are powerful antigen presenting cells that are being studied for use as anticancer vaccines. DCs modified by recombinant adenoviruses to express tumor-associated antigens may allow the presentation of longer antigenic sequences and improve antitumor immune responses. In an effort to translate this approach from animal models to the clinical setting, we compared adenoviral-mediated gene transfer in murine bone marrow-derived DCs (mDCs) and human peripheral blood monocyte-derived DCs (hDCs). hDCs were generated from peripheral blood monocytes in RPMI-1640 medium with 10% autologous human plasma supplemented with GM-CSF and IL-4. mDCs were generated from bone marrow cells obtained from female BALB/c mice cultured in RPMI-1640 medium with 10% fetal bovine serum and murine GM-CSF. hDCs expressed CD11c, CD40, CD80, CD86 and MHC class I and II, but did not express CD1a or CD83 indicating an immature DC phenotype. There was no significant change in expression of these markers following infection with an empty adenoviral vector (Ad.null). Stimulation of hDC with CD40L increased expression CD80, CD86, MHC class I and II, and CD83 indicating maturation. mDCs expressed CD11c, CD11b, CD80, CD86, CD40, CD54 and MHC class I and II but not CD8a indicating myeloid differentiation. Infection of mDCs with Ad.null increased the expression of CD80, CD86 and MHC class I and II. Forty-eight hours after infection (MOI = 30) with an adenovirus expressing yellow fluorescent protein (Ad.EYFP), 70% of mDCs expressed the transgene. At the same MOI, only 39% of hDCs expressed EYFP. However, at MOI = 100, more than 90% of the hDCs were positive for EYFP. Irradiated hDCs modified with Ad.null or Ad.Kras, a virus expressing non-signaling mutant human K-ras (G12V) induced greater 3H-thymidine uptake than unmodified hDCs in mixed culture with autologous lymphocytes. The greatest proliferation was induced using uninfected CD40L-matured hDCs. Similar results were seen with mDCs. Analysis by real-time RT-PCR comparing the relative levels of IL-12 p40 mRNA of DCs infected with Ad.null or Ad.Kras to unmodified DCs revealed increased IL-12 mRNA expression in the DCs treated with the adenoviruses. Stimulation of murine splenocytes with mDCs modified with recombinant adenoviral vectors, resulted in greater interferon (IFN)-gamma secretion than stimulation with unmodified DCs. IFN-gamma production by autologous lymphocytes cultured with hDCs was dramatically increased by modification of the hDC with recombinant adenoviral vectors compared to unmodified hDCs alone or hDCs matured with CD40L. Conclusions: (1) Gene transfer and high level gene expression can be accomplished in mouse and human DCs with recombinant adenoviral vectors, (2) Infection of mDCs and hDCs with recombinant adenoviruses enhanced expression of CD80 and CD86 co-stimulatory molecules, MHC class I and II, as well as IL-12, (3) hDC modified by recombinant adenoviruses stimulated lymphocyte proliferation and production of IFN-gamma, (4) Infection with adenovirus induced a Th1 cytokine expression pattern. Adenoviral-mediated gene transfer of tumor-associated antigens into DCs may offer a useful tool to stimulate the immune system against cancer.