The effects of bile acids on cytosolic free calcium ([Ca2+]i) were studied in single isolated rat hepatocyte couplets using a scanning laser cytometer and the fluorescent dye, indo-1. Cholestatic bile acids, chenodeoxycholate (CDC) and taurolithocholate (TLC) increased [Ca2+]i in a dose-dependent manner. Choleretic bile acids, tauroursodeoxycholate (TUDC) and taurocholate (TC), did not induce any change in [Ca2+]i except TC at very high doses. Treatment with TUDC added concomitantly with CDC or TLC significantly decreased the rise in [Ca2+]i induced by bile acids. These results, obtained with a polarized hepatocyte model that secretes bile, confirmed that cholestatic bile acids increase [Ca2+]i and showed that TUDC inhibits this phenomenon. These data are in agreement with a key role of intracellular calcium in cholestasis.