Calcium-mobilizing hormones and neurotransmitters are known to affect cell morphology and function including cell differentiation or division. In this study, we examined vasopressin (AVP)-induced morphological changes in a polarized system of rat hepatocytes. Light and electron microscope observations showed that AVP induced microvilli formation and a remodeling of the isolated hepatocyte F-actin submembrane cytoskeleton, these two events being correlated. We showed that these effects were rapid, reversible, observed at nanomolar AVP concentration and mediated by the V(1a) receptor. On polarized multicellular systems of hepatocytes, we observed a rapid reduction of the bile canaliculi lumen at the apical pole and micovilli formation at the basolateral domain with an enlarged F-actin cytoskeleton. Neither activation of protein kinase C nor A via phorbol ester or dibutyryl cAMP induced such rapid morphological changes, at variance with ionomycin, suggesting that AVP-induced intracellular calcium rise plays a crucial role in those effects. By using spectrofluorimetry and cytochemistry, we showed that calcium release from intracellular stores was involved in bile canaliculus contraction, while calcium entry from the extracellular space controlled microvilli formation. Taken together, AVP and calcium-mobilizing agonists differentially regulate physiological hepatocyte plasma membrane events at the basal and the apical domains via topographically specialized calcium-dependent mechanisms.