Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl^- absorption. However, the pathways for basolateral Cl^- exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl^- conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl^- conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl^- current that was inhibited by 4,4'-diisothiocyanotostilbene-2,2'-disulfonic acid (DIDS). The I-V relationship was nearly linear, and the halide selectivity was Cl^- > Br^- >> I^-. Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C and reducing pH. In contrast, increasing cell Ca²⁺ and inducing cell swelling had no effect. The basolateral Cl^- current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl^- currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl^- conductance that is not due to CFTR, ClC-2 or ClC-3. Its regulation by cAMP and protein kinase C signaling pathways suggest that coordinated regulation of Cl^- conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl^- movement.