A central function of cystic fibrosis transmembrane conductance regulator (CFTR)-expressing tissues is the secretion of fluid containing 100-140 mM HCO3-. High levels of HCO3- maintain secreted proteins such as mucins (all tissues) and digestive enzymes (pancreas) in a soluble and/or inactive state. HCO3- secretion is impaired in CF in all CFTR-expressing, HCO3--secreting tissues examined. The mechanism responsible for this critical problem in CF is unknown. Since a major component of HCO3- secretion in CFTR-expressing cells is mediated by the action of a Cl-/HCO3- exchanger (AE), in the present work we examined the regulation of AE activity by CFTR. In NIH 3T3 cells stably transfected with wild type CFTR and in HEK 293 cells expressing WT and several mutant CFTR, activation of CFTR by cAMP stimulated AE activity. Pharmacological and mutagenesis studies indicated that expression of CFTR in the plasma membrane, but not the Cl- conductive function of CFTR was required for activation of AE. Furthermore, mutations in NBD2 altered regulation of AE activity by CFTR independent of their effect on Cl- channel activity. At very high expression levels CFTR modified the sensitivity of AE to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate. The novel finding of regulation of Cl-/HCO3- exchange by CFTR reported here may have important physiological implications and explain, at least in part, the impaired HCO3- secretion in CF.