The aim was to explore whether biliary epithelial cells show muscarinic acetylcholine receptors and to investigate their role in ductular bile formation. In both, isolated rat biliary epithelial cells and Mz-Cha-1 cells, a biliary epithelial cell line, binding of [3H]N-methyl-scopolamine occurred with 0.718 ± 0.08 and 0.482 ± 0.05 fmol per 10(6) cells, respectively. To characterize the involved second messenger, intracellular Ca2+ levels were monitored by confocal microscopy. Stimulation of biliary epithelial cells with carbachol produced an increase in free cytosolic Ca2+ levels that declined to baseline values describing a sinusoidal oscillation curve. Increasing concentrations of the agonist decreased latency of the response and increased oscillation frequency. Similar results were obtained in Mz-Cha-1 cells. The intracellular Ca2+ originated from IP3 sensitive intracellular stores and from the extracellular medium. The Ca2+ response could partially be blocked by atropine and completely by pirenzepine, a specific muscarinic receptor-type M1 antagonist. The presence of M1 receptor messenger RNA (mRNA) in biliary epithelial cells was confirmed by reverse transcriptase polymerase chain reaction. In the isolated perfused guinea pig liver, a model with high ductular bile flow, carbachol induced a dose dependent decrease of bile flow by 79.6% ± 9.8% at 50 μmol/L carbachol (P < .001), without affecting perfusion pressure or biliary electrolyte concentrations. It is concluded that biliary epithelial cells express muscarinic acetylcholine receptors. Stimulation of this receptor leads to cholestasis. This could be because of changes in peribiliary permeability and/or inhibition of biliary epithelial cell secretory function.