Cystic fibrosis transmembrane conductance regulator (CFTR) channels conduct HCO₃^- in addition to Cl^- in airway epithelial cells. A defective bicarbonate-transporting function of CFTR may underlie the pathogenesis of CF disease. Here, we have investigated whether a bicarbonate-sensitive soluble adenylyl cyclase (sAC) is functionally coupled with CFTR and thus forms an autoregulatory mechanism for bicarbonate transport in human airway epithelial Calu-3 cells. Reverse transcriptase-polymerase chain reaction showed that transcripts of both full-length and truncated sACs are present in Calu-3 cells. Truncated sAC protein is the predominant, if not the only, isoform expressed in Calu-3 cells. Bicarbonate stimulated modest increase of cAMP production, and the increase was sensitive to 2-hydorxyestrodial (2-HE), a sAC inhibitor, but not to SQ22,536, a blocker of conventional transmembrane adenylyl cyclases. These results suggested that sAC is functional in Calu-3 cells. Adding 25 mM bicarbonate in the bath stimulated CFTR-mediated whole cell currents in the absence, but not in the presence, of 2-HE. In cell-attached membrane patches, 25 or 50 mM bicarbonate in the bath markedly increased Np_o of CFTR, and this activation was attenuated by 2-HE. These findings demonstrate that sAC signaling pathway is involved in the regulation of CFTR function in human airway epithelium, and thereby provides a link between the level of intracellular bicarbonate/CO₂ and the modulation of HCO₃^- -conductive CFTR function by cAMP/PKA.