To pursue the capacity of monkey dendritic cells (DC) to be modified by adenoviral vectors and present the encoded antigens, we generated DC from blood monocytes and infected them with recombinant adenoviruses encoding GFP reporter and SIVgag or nef genes. Recombinant, E1- and E3-deleted, adenoviruses could transfect immature DC to >90% efficiency. When differentiated in the presence of a maturation stimulus, the infected cells were identical to control uninfected DC in surface markers and potent stimulatory activity for the mixed leukocyte reaction. Recombinant adeno-SIVgag was comparable to vaccinia-gag in stimulating IFN-gamma-secreting CD8(+) T cells from PBMC of macaques vaccinated with SIV(mac239) Deltanef and challenged with pathogenic SIV or chimeric SIV/HIV. Small numbers of adeno-SIVgag-infected DC were sufficient to trigger specific ELISPOT responses by CD8(+) T cells from these animals. Some CD4(+) IFN-gamma-secreting cells were also found in the three of eight vaccinated animals with the highest CD8(+) responses. T cells from control animals did not respond to DC transfected with adeno-gag. Therefore recombinant adenoviruses efficiently transfect monkey DC in a nonperturbing fashion, and these DC efficiently present antigens to SIVgag immune CD8(+) T cells. These findings will allow autologous DC, expressing SIV genes with high efficiency, to be tested in vivo to achieve strong specific T cell immunity.