PKA-mediated phosphorylation of the regulatory domain (R-domain) plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately 6-fold that to PKA stimulation. In contrast, with hCFTR the response to PKC was about 90% of that to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted-cysteine-accessibility method we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately 5-fold increase in single-channel open probability (P_o), with a minor (ca. 30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in P_o. In both instances, there were no changes in the number of channels in the membrane. We speculate that, in other animals than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.