Recombinant poxviruses encoding tumor-associated antigens (TAA) are attractive as candidate cancer vaccines. Their effectiveness, however, will depend upon expression of the TAA in appropriate antigen-presenting cells. We have used a murine model in which the TAA is β-galactosidase (β-gal) and a panel of recombinant vaccinia viruses (rVV) in which β-gal was expressed under early or late promoters at levels that varied over 500-fold during productive infections in tissue culture cells. Remarkably, only those rVV employing early promoters were capable of prolonging the survival of mice bearing established tumors expressing the model TAA. Late promoters were ineffective regardless of their determined promoter strength. The best results were obtained when β-gal was regulated by a strong early promoter coupled to a strong late promoter. When a variety of cell types were infected with the panel of viruses in vitro, dendritic cells were found to express β-gal only under the control of the early promoters even though late promoters were intrinsically more active in other cell types. Furthermore, in a functional assay, dendritic cells infected in vitro with rVV encoding β-gal regulated by an early promoter activated β-gal-specific cytotoxic T lymphocytes, whereas similar rVV with a late promoter-regulated gene did not. These data indicate that promoter strength per se is not the most critical quality of a recombinant poxvirus-based tumor vaccine and that the use of promoters capable of driving the production of TAA in “professional” antigen presenting cells may be crucial.