Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC⑀ was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC⑀ interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC⑀ does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na