Cyclic AMP-mediated stimulation of hepatic bile acid uptake is associated with dephosphorylation and translocation of Ntcp to the plasma membrane. Although translocation of Ntcp may be facilitated by dephosphorylation, the mechanism of dephosphorylation is unknown. The ability of cAMP to translocate and dephosphorylate Ntcp is, in part, dependent on cAMP-mediated increases in cytosolic Ca²⁺, indicating that PP2B, a Ca²⁺/calmodulin dependent protein phosphatase, may be involved. Thus, we studied the role of PP2B using an inhibitor, cypermethrin (CM). Freshly isolated hepatocytes were pretreated with CM (1-5 nM) for 30 min followed by 15 min incubation with 10 µM CPT-cAMP. CM (5 nM) and FK506 (5 µM) inhibited cAMP-stimulated taurocholate (TC) uptake by 80 and 75%, respectively, without affecting basal TC uptake. CM also reversed cAMP-mediated Ntcp dephosphorylation and translocation to 80% and 15% of the basal level, respectively. Cyclic AMP stimulated PP2B activity by 60% and this effect was completely inhibited by 5 nM CM. PP2B dephosphorylated Ntcp immunoprecipitated from control, but not from cAMP-treated hepatocytes. The effect of CM was not due to any changes in cAMP-mediated increases in cytosolic [Ca²⁺] or decreases in MAPK (ERK1/2) activity. Taken together, these results suggest that cAMP dephosphorylates Ntcp by activating PP2B in hepatocytes and PP2B-mediated dephosphorylation of Ntcp may be involved in cAMP-mediated Ntcp translocation to the plasma membrane.