Hormonal control of the restoration of hepatocanalicular polarity in short-term cultured hepatocyte couplets was analyzed. One hour following isolation, couplets were unable to accumulate the fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), and showed a nonpolarized distribution of F-actin and mrp2 over the cell body. A progressive, time-dependent restoration of couplet-polarized function and morphology was reached after 4 hours of culture. Both dibutyryl cyclic adenosine monophosphate (DBcAMP) and the Ca2+-elevating compound, thapsigargin, accelerated restoration of normal couplet morphology and function. The DBcAMP-mediated stimulus was inhibited by the Ca2+ chelator, 1,2-bis-(o-aminophenoxy)-ethene-N,N,N′,N′-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM), but not by the protein kinase A (PKA) inhibitors, KT5720 or H89, suggesting that Ca2+ elevation rather than PKA activation is involved. N-(6-aminohexyl-5-chloro-1-napththalenesulfonamide (W-7), a calmodulin inhibitor, and the protein kinase C (PKC) activator, phorbol dibutyrate, inhibited both the basal and the DBcAMP-stimulated recovery of functional polarity, whereas staurosporine and Gö 6976, 2 PKC inhibitors, accelerated the basal recovery of polarized function. Disruption of the microtubule cytoskeleton by colchicine induced only minor changes under basal, but not under DBcAMP-stimulated, conditions. The Golgi complex disruptor, brefeldin A, significantly delayed, and the microfilament-disrupting agent, cytochalasin D, fully blocked, both processes. However, DBcAMP stimulated trafficking of vesicles containing CLF to the pericanalicular region under the last condition. Our results indicate that restoration of couplet polarity following isolation occurs via a Ca2+-calmodulin-mediated mechanism, which depends on microfilament, but not on microtubule integrity. A second pathway is activated by DBcAMP activation via Ca2+-calmodulin formation, whose requirements with respect to cytoskeletal components are opposite. PKC has a negative regulatory role in both pathways.