Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl− conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS·cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS·cm-2 and became anion selective, with the permeability sequence SCN− > NO3− > I− > Br− > Cl− > SO42− - gluconate - cyclamate. GaCl. l was not affected by the putative Cl− channel blockers Cu 2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl− channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl− channels were found. With ~ 100 mM Cl− in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of ~ 10 pS. The less frequent channel was outward rectifying with slope conductances of ~10 and 20 pS at -40 and 40 mV, respectively. The Cl− channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl− channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (~ 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS CIchannels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl− channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.