Cystic fibrosis (CF) is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The commonest CF mutation F508del-CFTR has both protein processing and channel gating defects. Small-molecule potentiators have been identified which target the gating defect of F508del-CFTR (1). However, little is known about their effects on other CF mutations. Here, we studied A561E-CFTR, the second commonest CF mutation in Portugal. Like F508del, A561E is a temperature-sensitive processing mutant located in the first nucleotide-binding domain (2), which severely disrupts channel gating (wild-type (wt), open probability (Po) ~ 0.42; A561E, Po ~ 0.06). To rescue the A561E gating defect with CFTR potentiators, we used BHK cells stably expressing A561E-CFTR and the iodide efflux assay; BHK-wt- and F508del-CFTR were studied as controls. We tested the potentiators PG-01 (P2), SF-03 (P3), UCCF-853 (P4), ΔF508act-02 (P5) and UCCF-180 (P9) (all from CF Foundation Therapeutics Compound Collection, and used at 10 µM in the presence of forskolin, 10 µM); genistein (50 µM) was used as a reference potentiator. At 37 °C, forskolin and genistein elicited a large transient iodide efflux from BHK-wt-CFTR, but were without effect on BHK-A561E- and BHK-F508del-CFTR. After low-temperature incubation for 24 h, genistein partially restored channel activity to A561E- and F508del-CFTR (84 ± 5, 74 ± 3%, respectively, means ± S.E.M, n = 4), relative to the response of BHK-wt-CFTR (termed, %wt-gen). All potentiators tested enhanced iodide efflux from both BHK-A561E- and F508del-CFTR (n = 4, P < 0.05, unpaired T-test versus vehicle DMSO). However, none of them restored channel activity to wt-CFTR levels. For BHK-A561E-CFTR, the rank order of potentiation was P4 ≥ P9 ≥ P2 > P5 ≥ P3 (51 ± 3, 38 ± 5, 33 ± 2, 15 ± 2, 14 ± 1, respectively, %wt-gen, means ± S.E.M, n = 4) and for BHK-F508del-CFTR, it was P2 ≥ P5 ≥ P4 ≥ P3 ≥ P9 (22 ± 2, 20 ± 1, 19 ± 3, 15 ± 3, 10 ± 1, respectively, %wt-gen, means ± S.E.M, n = 4). Examination of the concentration-response relationship for P9 revealed that the half maximal effective concentration (EC50) for potentiation of wt- and A561E-CFTR was similar (~ 5 µM), whereas that of F508del-CFTR was higher (~ 10 µM). In conclusion, our data suggest that all tested small molecules that potentiate F508del-CFTR also enhance A561E-CFTR channel function, and the overall potentiating effects are better in A561E-CFTR expressing cells. However, variations in the rank order and also in P9 affinity may indicate that these two-mutant CFTRs disrupt channel gating through distinct mechanisms.