Canalicular bile is formed by the osmotic filtration of water in response to osmotic gradients generated by active transport at the apical and basolateral plasma membrane domains of hepatocytes. We recently demonstrated that mixed plasma membrane fractions isolated from rat hepatocyte couplets contain lipid microdomains ("rafts") enriched in cholesterol and sphingolipids and AQP8 and 9. We isolated lipid microdomains from hepatocyte apical and basolateral plasma membrane domains using Triton X-100 as detergent, and characterized their lipid and protein composition. A Triton-insoluble band ("raft fraction") at the 5%/30% sucrose interface in both apical and basolateral fractions was enriched for alkaline phosphatase (apical) and Na/K ATPase (basolateral) and was negative for amino peptidase-N. This detergent-insoluble band was also positive for caveolin-1 (a "raft" associated protein) and negative for clathrin (a "raft" negative protein). Lipid analysis showed that, the Triton-insoluble fraction was highly enriched in cholesterol and sphingolipids. Immunofluorescence staining on hepatocyte couplets for both caveolin-1 and cholera toxin B showed a punctate distribution on both the apical and basolateral plasma membranes, consistent with localized membrane microdomains. Dot blot analysis showed that the "raft" associated ganglioside GM1 was enriched in the detergent-insoluble fraction both domains. Furthermore, exposure of isolated hepatocytes to glucagon, a choleretic agonist, significantly increased the expression of AQP8 associated with the apical microdomain fractions but had no effect on AQP9 expression in the basolateral microdomain fractions. In conclusion, "rafts" represent target microdomains for exocytic insertion and retrieval of "flux proteins", including AQPs, involved in canalicular bile secretion.