The most common cystic fibrosis (CF) mutation, F508del, causes defective cystic fibrosis transmembrane conductance regulator (CFTR) processing, decreased protein stability and aberrant Cl- channel gating. To overcome these defects, small-molecules termed CFTR correctors and potentiators have been developed. Here, we investigate the stability of F508del-CFTR in excised membrane patches, intact cells and polarised epithelia using patch-clamp, iodide efflux and Ussing chamber assays and cells expressing recombinant CFTR (1, 2). To promote the cell surface expression of F508del-CFTR, F508del-CFTR expressing cells were incubated at 27 °C prior to use. In excised membrane patches, wild-type CFTR exhibited robust channel activity at 37 °C. At 23 °C, F508del-CFTR channel activity was sustained at a very low level (n = 5). At 37 °C, the open probability (Po) of F508del-CFTR was initially greater than that at 23 °C, but still much lower than wild-type and it declined rapidly to zero within 7 minutes (n = 5). To explore further the cell membrane stability of F508del-CFTR, we used iodide efflux to study the behaviour of large numbers of mutant Cl- channels in intact cells. Following incubation at 27 °C for 24 h to deliver F508del-CFTR to the cell membrane, cells were returned to 37 °C and the magnitude of CFTR-mediated iodide efflux measured over an 8 h period. Wild-type CFTR generated iodide efflux was stable over time, whereas that elicited by F508del-CFTR decreased steadily (t½ ∼7 h; n = 4 – 6). Employing a similar protocol to that used for iodide efflux, we observed comparable results with recombinant Fischer rat thyroid (FRT) epithelia expressing F508del-CFTR; F508del-CFTR-mediated transepithelial Cl- currents decreased steadily (t½ ∼6.5 h; n = 4 – 11). To begin to explore the impact of CFTR modulators on F508del-CFTR stability, we used the dual-acting small-molecule CFFT-004, which acts as both a CFTR corrector and potentiator. When tested as either a corrector or a potentiator, CFFT-004 (5 μM) increased the initial Po of F508del-CFTR in excised membrane patches at 37 °C without conferring stability on F508del-CFTR; the Po of F508del-CFTR declined to zero within 7 minutes (n = 4 – 7). Interestingly, combining low temperature incubation with CFFT-004 (5 μM)-correction, increased greatly the initial magnitude of F508del-CFTR-mediated transepithelial Cl- current (untreated, 54 ± 4 µA cm-2; CFFT-004-corrected, 89 ± 12 μA cm-2; n = 11, Students t-test, P < 0.01), but accelerated Cl- current decline (t½ ∼4.5 h; n = 4 – 11). In conclusion, F508del-CFTR stability can be evaluated functionally at the level of single molecules, intact cells and polarised epithelia to provide insight into the action of CFTR modulators.