The fluidity of basolateral and canalicular rat liver plasma membranes was compared with respect to their response to the membrane perturbants ethanol and calcium. The relation between membrane fluidity and taurocholate transport, a liver plasma membrane function mediated by carrier proteins, was also examined. Membrane fluidity was measured by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as a probe. Uptake of [3H]taurocholate into basolateral rat liver plasma membrane and canalicular rat liver plasma membrane vesicles was measured by a rapid Millipore filtration technique. Anisotropy values were found to be significantly lower for the basolateral rat liver plasma membrane (0.2287 +/- 0.0014) than for the canalicular rat liver plasma membrane (0.2612 +/- 0.0012), indicating that basolateral rat liver plasma membranes are more fluid than canalicular rat liver plasma membranes. Ethanol produced a concentration-dependent effect on lipid fluidity and inhibition of taurocholate uptake, in both membrane preparations. Pretreatment of the membrane with calcium increased the rigidity of both membrane preparations. However, the change in the anisotropy with calcium was only slight in the more rigid canalicular rat liver plasma membrane, while the change in anisotropy was greater and associated with a decrease in taurocholate uptake in the basolateral rat liver plasma membrane. Both the effects of ethanol and calcium were more pronounced in basolateral rat liver plasma membrane than in canalicular rat liver plasma membrane. These results indicate that the fluid state of the hydrophobic bilayer of liver plasma membrane lipids play an important role in regulating bile acid transport in both sinusoidal and canalicular domains.