CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity.