Recombinant poxviruses expressing tumor-associated antigens (TAAs) are currently being evaluated in clinical trials as an approach to treat various cancers. We have previously generated poxviral vectors expressing a TAA and a TRIad of COstimulatory Molecules (B7-1, ICAM-1, and LFA-3; TRICOM) as transgenes, including replication competent recombinant vaccinia (rV) or replication-defective modified vaccinia Ankara (MVA), to prime tumor-specific immune responses, and a replication-defective recombinant fowlpox (rF) to boost these responses. MVA is a potentially safer, replication-defective form of vaccinia virus with unique immunostimulatory properties that could make it a superior priming vaccine. Here, an MVA vector encoding a tumor antigen (CEA) and TRICOM was utilized (rMVA). A single rMVA-CEA/TRICOM vaccination induced greater expression of several serum cytokines associated with enhanced T-cell immunity than that seen with vaccinia. We hypothesized that this effect might "precondition" the vaccination site for a more effective boost. An rMVA-CEA/TRICOM prime followed 7 days later (but not 30 days later) by an rF-CEA/TRICOM boost at the same injection site (but not at a distal site) induced more potent CEA-specific T-cell responses, and superior CEA-specific immunity and antitumor activity, than rV-CEA/TRICOM followed by rF-CEA/TRICOM. This preconditioning effect was also observed using a heterologous antigen model, where priming with rMVA-CEA/TRICOM followed 7 days later by rF-LacZ/TRICOM enhanced beta-gal-specific immunity compared to rF-LacZ/TRICOM only. The studies reported here show for the first time that priming with rMVA followed 7 days later by an rF boost at the same injection site, versus a distal site, generates superior tumor-specific immunity and antitumor activity.