Peribiliary glands (PBGs) are clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts (EHBDs). Though their function is largely undefined, they may represent a stem cell niche. Here, we hypothesized that PBGs are populated by mature and undifferentiated cells capable of proliferation in pathological states. To address this hypothesis, we developed a novel whole-mount immunostaining assay that preserves the anatomical integrity of EHBDs coupled with confocal microscopy and found that PBGs populate the entire length of the extrahepatic biliary tract, except the gallbladder. Notably, in addition to the typical position of PBGs adjacent to the duct mucosa, PBGs elongate and form intricate intramural epithelial networks that communicate between different segments of the bile duct mucosa. Network formation begins where the cystic duct combines with hepatic ducts to form the common bile duct (CBD) and continues along the CBD. Cells of PBGs and the peribiliary network stain positively for α-tubulin, mucins, and chromogranin A, as well as for endoderm transcription factors SRY (sex determining region Y)-box 17 and pancreatic and duodenal homeobox 1, and proliferate robustly subsequent to duct injury induced by virus infection and bile duct ligation. Conclusion: PBGs form elaborate epithelial networks within the walls of EHBDs, contain cells of mature and immature phenotypes, and proliferate in response to bile duct injury. The anatomical organization of the epithelial network in tubules and the link with PBGs support an expanded cellular reservoir with the potential to restore the integrity and function of the bile duct mucosa in diseased states. (Hepatology 2013;58:1486–1496)