Canalicular transport via the bile salt export pump (Bsep) represents the rate‐controlling step in taurocholate excretion, whose capacity is under osmotic control. The short‐term effects of anisoosmolarity and Ca2+‐withdrawal on the localization of Bsep and the tight junction proteins Zo‐1 and occludin were studied in perfused rat liver by immunohistochemistry, confocal microscopy, and densitometry. Under normoosmotic conditions, Bsep was found in the canalicular membrane and showed a punctate intracellular localization. Hypoosmolarity resulted in the translocation of intracellular Bsep to the canalicular membrane, whereas hyperosmolarity induced a retrieval of Bsep. Following hyperosmolar retrieval of Bsep and multidrug resistance protein 2 (Mrp2) from the canalicular membrane, in the putative intracellular vesicles Bsep and Mrp2 colocalized in 15% of these vesicles, whereas 85% stained either positive for Bsep (61%) or Mrp2 (24%). Anisotonicity had no effect on the linear staining patterns of occludin and Zo‐1, indicating no increase in paracellular permeability. Omission of calcium produced cholestasis characterized by a disruption of occludin, whereas the localization of Zo‐1, Bsep, and Mrp2 remained unaffected. It is concluded (1) that hyperosmolarity induces retrieval of Bsep from the canalicular membrane, which correlates to cholestasis. Hypoosmolarity leads to choleresis accompanied by a rapid recruitment of intracellular Bsep to the canalicular membrane. (2) Bsep‐ and Mrp2‐specific vesicles participate in the short‐term osmoregulation of canalicular secretion, however, a cause‐effect relationship between bile salt excretion and transporter localization remains to be established. (3) Ca2+‐depletion induces cholestasis by disruption of occludin‐determined tight junctional permeability, whereas internalization of canalicular transporters play a minor role.