Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study we developed a bicistronic construct called R-Split CFTR which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel, and co-expression of the third fragment corresponding to the missing RD restores regulation by PKA and this is associated with dramatically enhanced binding of phosphorylated RD. In the present study we examine the effect of PKC phosphorylation on this PKA induced interaction. We report here that PKC alone enhances association of the RD with R-Split CFTR, and that binding is further enhanced when the RD is phosphorylated by both kinases. Mutating all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but it abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. Basal activity of R-Split/7CA-RD channels was similar to that of R-Split/WT-RD channels whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.