The activity of the human CFTR channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus CFTR (XCFTR) is more responsive to PKC than to PKA stimulation. Here, we report an investigation of the interaction between the two kinases in XCFTR. We expressed in XFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of the activation following PKC stimulation was about eight-fold that without pretreatment with PKC agonist. Human CFTR (hCFTR), expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied the single-channel properties, and found that the single-channel open probability with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC pre-activation, and that the single-channel conductance was increased by ca. 22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR/hCFTR chimeras, expressed them in Xenopus oocytes and tested them electrophysiologically. Two chimeras (hCFTR N-terminal region or R-domain in XCFTR) showed a significant decrease in the potentiation. In the chimera in which XCFTR NBD2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that the potentiation by PKC involves a large increase in single-channel open probability (with a small change in single-channel conductance) without CFTR channel insertion into the plasma membrane, that the XCFTR NBD2 is necessary, but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains.