The most common cystic fibrosis mutation, F508del, disrupts cystic fibrosis transmembrane conductance regulator (CFTR) function by impairing protein processing, plasma membrane stability and channel gating. Small molecules called correctors (e.g. lumacaftor [VX-809]) and potentiators (e.g. ivacaftor [VX-770]) have been developed to rescue F508del-CFTR. In a recent clinical trial [1], VX-770 + VX-809 combination therapy achieved milder than expected therapeutic benefit, possibly due to chronic VX-770 (cVX-770) destabilising VX-809-rescued F508del-CFTR [2,3]. Here, we use single-channel recording and Ussing chamber studies to investigate the effects of cVX-770 on VX-809-rescued F508del-CFTR. Single-channel data from BHK cells expressing F508del-CFTR (see Ref 4) revealed that acute treatment of VX-809-rescued F508del-CFTR with VX-770 (10 µM) improved greatly open probability (Po) and mildly stabilised F508del-CFTR activity. At the single-channel level, this stabilising effect was enhanced markedly by cVX-770 treatment (1 µM, 24 h). However, when F508del-CFTR Cl- currents were studied in excised membrane patches treated with VX-809 (3 µM, 24 h) and cVX-770 (1 µM, 24 h) the majority of current declined rapidly to leave a small population of highly active channels, similar to those observed in single-channel recordings. We interpret this result to suggest that there are two populations of F508del-CFTR Cl- channels one of which is stabilised by cVX-770 treatment. To understand how cVX-770 impacts transepithelial Cl- transport, we performed Ussing chamber studies on Fischer rat thyroid (FRT) epithelia expressing F508del-CFTR. After 24 h treatment with VX-809 (3 µM) and VX-770 (1 µM), we recorded the F508del-CFTR-mediated short-circuit current (Isc) in the presence of a basolateral to apical Cl- gradient. Chronically administered VX-809 and VX-770 enhanced greatly F508del-CFTR Isc compared to untreated controls, reaching ~25% of wild-type levels. To determine the impact of VX-770 administration on F508del-CFTR functional stability, small molecules were washed from epithelia and Isc recordings made at time (t) = 0, 2, 4 and 6 h after washout. In epithelia treated with VX-809 alone, Isc rapidly declined to untreated levels by 2 h, suggesting that in the absence of VX-809 little newly synthesised CFTR was trafficked to the apical membrane. While VX-809 + cVX-770 did not improve Isc at t = 0 h compared to VX-809 treatment alone, it did improve F508del-CFTR functional stability consistent with single-channel data. We conclude that chronic co-administration of VX-809 and VX-770 appears to rescue a sub-population of F508del-CFTR Cl- channels in recombinant cells. Further work is required to identify and characterise these channels.