Current research in gene delivery to the liver is focused on the intravenous, intraarterial, intraportal, or intratumoral route. Another possible route for gene delivery is via the common bile duct through endoscopic retrograde cholangiopancreatography (ERCP). Whether bile and pancreatic juice have any effect on gene delivery is not established. To evaluate the effect of bile and pancreatic juice on adenoviral-mediated gene delivery, liver and pancreatic cell lines were infected with a recombinant adenovirus expressing an E. coli beta-galactosidase gene under the control of a cytomegalovirus promoter (rAdCMVpLacZ) in the absence or presence of various concentrations of bile and pancreatic juice. The proportion of cells infected was evaluated through X-gal staining. The toxicity of bile and pancreatic juice was also evaluated through cell morphology and detachment. Bile appeared to induce significant cytotoxicity in HepG2 and Huh7 cells (50% viability with 15 min of incubation). Neither bile nor pancreatic juice affected transgene expression. In the absence of bile/pancreatic juice, HepG2 (15-25%) and PANC-1 cells (10-18%) were less susceptible to rAdCMVpLacZ compared to Huh7 cells (75-84%, vs HepG2, P < 0.001) and BxPc-3 (82-95%, vs PANC-1, P < 0.001) at a multiplicity of infection (MOI) of 5. Bile reduced the transduction efficiency, but 5-10% HepG2 and 5-42% of Huh7 cells were still transduced in the presence of 80% bile for up to 10 min. Adenoviral-mediated gene delivery was reduced in the presence of pancreatic juice with a low multiplicity of infection (MOI of 5), but this effect was negated with an MOI of 50. These data provide encouragement to develop adenoviral-mediated gene delivery through ERCP.