Dendritic cells (DC) are potent antigen-presenting cells (APC). Ongoing preclinical and clinical studies exploit this capacity for the immunotherapy of tumors. We tested vaccinia virus (VV) as a vector to transduce human DC. Immature and mature DC were prepared from blood monocytes and infected with (1) recombinant VV expressing GFP to analyze infection rates, virus replication in DC and the effect of infection on DC phenotype and (2) recombinant VV expressing beta-galactosidase (βGAL) under the control of viral early, intermediate and late promoters to analyze the poxvirus-driven gene expression. While the infection rate in DC was comparable to a permissive fibroblast cell line, viral βGAL gene expression was limited to early promoters. Genes under the control of virus late promoters were not expressed by VV in DC, indicating an abortive infection. VV infection selectively reduced the surface expression of the costimulatory molecule CD80 and the DC maturation marker CD83 on mature DC while other surface molecules including CD86 and MHC remained unchanged. In line with this finding, there was a pronounced reduction in the capacity of VV-infected DC to stimulate allogeneic or autologous T cells in mixed lymphocyte reactions. Furthermore, VV infection inhibited the maturation of immature DC after exposure to proinflammatory cytokines. These results indicate that VV-derived vectors may have complex effects on their target cells. In the case of DC used for immunotherapy, this may be detrimental to their function as potent APC and particularly their capacity to activate T helper cells.