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Background Biliary atresia (BA) is the most common indication for liver transplantation in children. The experimental model of BA, induced by rotavirus infection in neonatal mice, has been widely used to investigate the inflammatory aspects of this disease. We investigated the kinetics and the localization of the viral infection in this murine model. Methods In this study 399 animals were employed for a detailed investigation of rhesus rotavirus (RRV)-induced BA. RRV kinetics was analyzed by rtPCR and its (sub) cellular localization investigated using whole mounts which were further processed for confocal and electron microscopy. Results The BA mouse model resulted in up to 100% induction of atresia following RRV injection. The kinetics of RRV infection differed between liver and extrahepatic bile ducts. While the virus peak up to day 10 postinfection was similar in both organs, the virus remained detectable in extrahepatic bile duct cells up to day 21. Interestingly, RRV particles were localized not only in cholangiocytes but also in cells of the subepithelial layers, potentially macrophages. Conclusions RRV remains present in the extrahepatic bile duct cells after an initial virus peak. Viral particles were detected in subepithelial cells in contrast to the described tropism toward cholangiocytes.