Background: The full realization of the therapeutic potential of conditionally replicative adenoviruses (CRAds) in the field of pancreatic cancer has been hindered by limited tumor transduction and suboptimal replication control. Methods: We optimized infectivity enhancements and tumor-specific promoters (tsps) for pancreatic cancer. Infectivity was enhanced both by incorporating an RGD motif and by substituting the knob region with Ad serotype 3 knob (Ad5/Ad3). An optimized CRAd was tested in an orthotopic pancreatic cancer model by systemic administration. Results: Among a panel of 8 tsps, the 1.5-kb cyclooxygenase-2 (Cox-2L) promoter profile was most advantageous in the pancreatic cancer cell lines, whereas 4 more promoters were also promising. An infectivity-enhanced Ad5/Ad3 CRAd controlled with Cox-2L promoter was found to safely exhibit replication within a tumor in this model and was found to suppress tumor growth after systemic delivery. Conclusions: The infectivity-enhanced, promoter-controlled CRAd promises useful clinical applications for pancreatic cancer gene therapy. © 2008 Elsevier Inc. All rights reserved.