We studied the effect of adding chemotherapy or vector targeted chemotherapy to the administration of an Ad-sig-hMUC-1/ecdCD40L adenoviral vector prime-hMUC-1/ecdCD40L protein boost cancer vaccine (designated hMUC-1/ecdCD40L VPPP vaccine), which were administered to test mice 10 days following subcutaneous (s.c.) inoculation of 500,000 Lewis Lung Carcinoma cells, at a time when the average volume of the s.c. tumors was 50 cu mm. The survival of hMUC-1/ecdCD40L VPPP vaccine-treated mice was twice as long as untreated mice. Addition of vector-targeted chemotherapy (AdCMVCDIRESE1A/5FC) to the hMUC-1/ecdCD40L VPPP vaccine 10 days after tumor inoculation significantly (P=0.0062) prolonged the survival of the test mice over administration of the hMUC-1/ecdCD40L VPPP vaccine alone or the control mice (P<0.00001). Interestingly, the combination of the AdCMVCDIRESE1A/5FC vector-targeted chemotherapy to the hMUC-1/ecdCD40L VPPP vaccine decreased the levels of CD44(+)CD24⁻ cells in s.c. deposits of the human MUC-1-positive Lewis Lung Cancer cell line (LL2/LL1hMUC-1) by 20 fold. These results suggest that the addition of vector-targeted chemotherapy to an adenoviral-based cancer vaccine is a strategy that deserves further testing.