Due to devastating prognosis, novel therapies are needed for pancreatic cancer. We are in preparation for a human clinical trial of a conditionally replicative adenovirus (CRAd) we developed. While most patients in the target population are receiving either gemcitabine or 5-fluorouracil chemotherapy, the combination with CRAd has not yet been studied. This study was designed to evaluate combination therapies with CRAd and current standard chemotherapies in pancreatic cancer. When the combination therapy was tested in vitro, gemcitabine pretreatment showed a synergistic effect in two out of four cell lines whereas CRAd followed by gemcitabine exhibited a synergistic effect in one cell line. With 5-fluorouracil, pretreatment with 5-fluorouracil produced a synergistic effect in three cell lines whereas post-treatment was synergistic in only one cell line. These effects were not fully explained by either induction of cyclooxygenase (Cox) 2 activity or adenoviral receptors with chemotherapeutics. In in vivo analyses with Hs766T xenograft, 5-fluorouracil slightly improved the CRAd antitumor effect but it was not significant. Pretreatment with gemcitabine embodied a significant tumor reduction compared with single therapy with gemcitabine. The most significant antitumor effect occurred when tumors were treated with 5/3COX2CRAdF and subsequent gemcitabine (P = 0.001 vs gemcitabine alone, P = 0.012 vs 5/3COX2CRAdF alone) at day 12. In MIA Paca-2, pretreatments with either 5-fluorouracil or gemcitabine improved the CRAd therapeutic effect when administered before CRAd injection (P = 0.03 and P = 0.01, respectively). These experiments indicate the possible benefit of combination therapies, and thus it is not necessary to interrupt chemotherapeutics when receiving CRAd therapy. (Cancer Sci 2009).