In guinea pig gallbladder epithelium, a secretion of fluid, secondary to an electrogenic secretion of Cl− and HCO−3, is elicited in the presence of a high intracellular concentration of adenosine 3′-5′-cyclic monophosphate (cAMP). The aim of this study was to analyze the effects of secretagogues on the activity of anionic channels in isolated epithelial cells using the patch-clamp technique and measuring the electrical potential difference of the cellular membrane (pdcm). In cell-attached configuration, with the microelectrode filled with a solution of N-methylglucamine–Cl, or in inside-out configuration (symmetrical solution), it was possible to demonstrate the presence of an 18-pS Cl− channel with linear current/voltage (I/V) relationship and voltage independence; this channel is not activated by cAMP (cell-attached configuration). In inside-out configuration (symmetrical solution), another anionic channel with a conductance of 2.8 pS, voltage independence, and a linear I/V relationship was also identified. This channel was stimulated by cAMP (cell-attached configuration) and by PKA + ATP + cAMP (inside-out configuration). The channel was inhibited by NPPB (10−5 M), but not by other anionic inhibitors. Measurements of the pdcm value suggested that in isolated cells, as in whole tissue, cAMP activates conductance for both Cl− and HCO−3. The selectivity of the channel was gluconate < SO2−4 < Cl− < Br− < I− < HCO−3 < SCN− and the PHCO3/PCl was 2.6. Some features of the channel resemble those of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and RT-PCR performed on mRNA from isolated epithelial cells detected the presence of a CFTR homologue mRNA. The results obtained indicate that this channel is responsible for the HCO−3 conductance activated by cAMP.