BACKGROUND & AIMS: The copper transporter ATP7B plays a central role in the elimination of excess copper by the liver into the bile, yet the site of its action remains controversial. The studies reported here examine the correspondence between the site of ATP7B action and distribution and the pathways of copper disposal by the liver. METHODS: Microscopy and cell fractionation studies of polarized Can 10 cells forming long-branched bile canaliculi have been used to study the cellular distribution of ATP7B. Copper excretion into the bile was studied in perfused rat liver. RESULTS: Copper excess provokes a massive download of the ATP7B retained in the trans-Golgi network into the bile canalicular membrane. Furthermore, a stable ATP7B pool is localized to the tight junctions that seal the bile canaliculi. The profile of Cu(64) excretion into the bile by isolated rat livers perfused under one-pass conditions provides evidence of copper excretion by 2 separate mechanisms, transcytosis across the hepatocyte and paracellular transport throughout the tight junctions. CONCLUSIONS: Whereas the ATP7B retained in the trans-Golgi-network is massively translocated to the bile canalicular membrane in response to increased copper levels, a pool of ATP7B associated with the tight junctions remains stable. In situ studies indicate that copper is excreted into the bile by 2 separate pathways. The results are discussed in the frame of the normal and impeded excretion of copper into the bile.