1. A method is described for the detection of CFTR chloride channel openers (ClCOs) and blockers. Murine colonic epithelia were used throughout, but the method is applicable to other epithelia and biopsy material. 2. The principle was to render the epithelial basolateral membranes electrically transparent so that the apical membrane alone could be voltage clamped. This was achieved by potassium depolarization on the basolateral side. Imposition of an apical to basolateral chloride gradient allowed the effects of ClCOs on an outward chloride current and on apical membrane conductance to be measured. 3. 1-ethyl-2-benzimidazolone (EBIO), forskolin, chlorzoxazone, and genistein all showed ClCO activity. In cystic fibrosis (CF) epithelia, either from CF null or CF Delta F508 mice, EBIO showed only a minor effect, indicating that CFTR was the target in wild type tissues. 4. 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was shown to block CFTR chloride channels. The blockade was pH and voltage-dependent and indicated that while the charged form was the active moiety, movement into the cell depended on the unionized drug. It is concluded NPPB blocks CFTR from the cytosolic side and that the agent preferentially blocks at potentials opposing the inflow of chloride ions. No significant blockade was seen with either N-phenylanthranilic acid (DPC) or with glibenclamide, under standard conditions. 5. The method described can be used to examine compounds reported to increase the trafficking of Delta F508 CFTR to the membrane or those capable of opening Delta F508 CFTR chloride channels and to differentiate between them. Further, the method distinguishes between chloride channel openers and those acting indirectly to increase the flux through CFTR chloride channels by indirect means, for example, hyperpolarization.