Cystic fibrosis (CF) is caused by mutations that disrupt the expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. To develop transformational drug therapies for CF, small-molecules have been identified that rescue the expression (CFTR correctors) and function (CFTR potentiators) of mutant Cl- channels. Interestingly, some small-molecules have been identified with both CFTR corrector and potentiator activity (CFTR corrector-potentiators or dual-acting compounds; 1). Here, we investigate the action of the dual-acting small-molecule CFFT-004 (WO2010/068863) on F508del-CFTR using BHK cells stably expressing F508del-CFTR and the iodide efflux technique. As controls, we studied wild-type CFTR and A561E, a CF mutant with a similar clinical phenotype and mechanism of dysfunction, but different location in CFTR (2) and used the CFTR corrector corr-4a, and the CFTR potentiator genistein. We began by testing the action of CFFT-004 as a CFTR corrector. Incubation of BHK cells expressing F508del-CFTR with CFFT-004 (5 μM) or corr-4a (10 μM) for 24 h at 37 °C, but not the vehicle (DMSO; 0.05% vv-1), restored CFTR-mediated iodide efflux to F508del-CFTR that were similar in magnitude to those achieved by low temperature (27 °C) incubation for 24 h (n = 4 – 6). By contrast, treatment of cells expressing A561E with CFFT-004 (5 μM) or corr-4a (10 μM) for 24 h at 37 °C failed to confer CFTR-mediated iodide efflux on A561E-CFTR, whereas low temperature incubation restored some CFTR-mediated iodide efflux (n = 5). Next, we tested the action of CFTR-004 as a CFTR potentiator. When low temperature-rescued F508del-CFTR was treated with forskolin (10 μM) and CFFT-004 (5 μM), the magnitude of CFTR-mediated efflux was similar to that elicited by forskolin (10 μM) and genistein (50 μM; n = 5). Interestingly, treatment of low temperature-rescued A561E-CFTR with CFFT-004 (5 μM) generated an efflux of iodide comparable to that observed with low temperature-rescued F508del-CFTR treated with CFFT-004 (5 μM; n = 5). Finally, we tested CFFT-004 for dual-activity by incubating cells expressing F508del- and A561E-CFTR with CFFT-004 (5 μM) for 24 h at 37 °C before stimulating iodide efflux with forskolin (10 μM) and CFFT-004 (5 μM). CFFT-004 both corrected and potentiated F508del-CFTR, whereas it was without effect on A561E-CFTR consistent with the results of correction testing (n = 4 – 6). We conclude that CFFT-004 acts as a dual-acting molecule to rescue, albeit partially, the expression and function of F508del-CFTR. Based on our present and previous (2) data, we also conclude that the processing defect of A561E-CFTR is distinct from that of F508del-CFTR and different CFTR correctors will likely be required to delivery A561E-CFTR to the cell surface.