The activity of the CFTR Cl^– channel is dependent on its phosphorylation status set by kinases and phosphatases. We report here that protein phosphatase 2B (PP2B) and protein kinase C (PKC) are potential regulators of the cystic fibrosis conductance regulator (CFTR). Treating CFTR-expressing 3T3 cells with either of the two specific PP2B blockers cyclosporin A (CsA, 1 μM) or deltamethrin (DM, 30 nM) caused rapid activation of CFTR in cell-attached patches. As determined by noise analysis of multi channel patches, DM- or CsA-activated CFTR displayed gating kinetics comparable to those of forskolin-activated CFTR. After activation of CFTR by blocking PP2B, CFTR still inactivated. CFTR-mediated currents were, on average, 6.1 times larger when cells were stimulated by forskolin during PP2B block compared to stimulation by forskolin alone. This suggests that, in CFTR-expressing 3T3 cells, a phosphorylation site of CFTR is regulated by cellular PKA, PP2B and another phosphatase. However, in the epithelial cell lines Calu-3 and HT-29/B6, CsA and DM had no effect on CFTR activity in both cell-attached patch-clamp and transepithelial experiments. In contrast, when exogenous PP2B was added to patches excised from 3T3 or Calu-3 cells, PKA-activated CFTR currents were quickly inactivated. This indicates that free exogenous PP2B can inactivate CFTR in patches from both cell types. We propose that in order to regulate CFTR in an intact cell, PP2B may require a selective subcellular localization to become active. When excised patches were PKC-phosphorylated, the gating kinetics of CFTR were significantly different from those of PKA-phosphorylated CFTR. Addition of PP2B also inactivated PKC-activated CFTR showing the indiscriminate dephosphorylation of different phosphorylation sites by PP2B.