The cystic fibrosis gene encodes a chloride channel CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A-dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH2-terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail which is a potential target for physiologic and pharmacologic modulators of this ion channel.