Our laboratory has created an Ad-sig-TAA/ecdCD40L vaccine platform designed to activate dendritic cells (DCs). Two subcutaneous (s.c.) injections of the TAA/ecdCD40L protein following the s.c. injection of the Ad-sig-TAA/ecdCD40L vector (TAA/ecdCD40L VPP vaccine) further increases the levels of the tumor-associated antigen (TAA)-specific CD8 effector T cells induced by the vector. We tested the combined effect of chemotherapy-induced destruction of tumor cells and TAA/ecdCD40L VPP vaccine which further increases the levels of TAA available to the DCs at the time of vaccination. The chemotherapy was delivered selectively to the tumor cells using intratumoral (i.t.) injection of the AdCDIRESE1A vector followed by intraperitoneal (i.p.) 5-fluorocytosine (5FC). The 5-fluorouracil (5FU) produced in the vector infected the tumor cells, destroys them and releases the TAA for processing and presentation by the DCs. This mode of delivery spares the TAA CD8 effector T cells from the destructive effect of the 5FU when their proliferation is induced by the vaccine. Test mice treated with both the s.c. administered TAA/ecdCD40L VPP vaccine and the AdCDIRESE1A/5FC chemosensitization vector had the smallest tumor volumes and survived longer than mice treated with either of these agents alone ( P < 0.001).