Some amino acids in the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) are the site of more than one CFTR variant that cause the genetic disease cystic fibrosis.  Here, we investigate the function of two rare missense substitutions of the serine residue at codon 1159 in the pore-lining twelfth transmembrane segment, S1159F and S1159P and their response to the clinically-approved CFTR potentiator ivacaftor.  To study the single-channel behaviour of CFTR, we used excised inside-out membrane patches from CHO cells transiently expressing the CFTR variants that were incubated at 27 °C for 7-12 days to enhance channel trafficking to the plasma membrane.  S1159F- and S1159P-CFTR formed Cl^– channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37 °C.  At –50 mV in the presence of a large Cl^– concentration gradient ([Cl^–]_int, 147 mM; [Cl^–]_ext, 10 mM), ATP (1 mM) and protein kinase A (75 nM), both variants modestly reduced single-channel current amplitude (i), but severely decreased open probability (P_o) (S1159F, n = 21; S1159P, n = 25).  Ivacaftor (10 – 100 nM) doubled the P_o of both CFTR variants, but did not restore P_o values to wild-type levels (S1159F, n = 3–5; S1159P, n = 8–9).  Interestingly, higher concentrations of ivacaftor (0.5 – 1 µM) had little effect on S1159P-CFTR, but reduced the i of S1159F-CFTR (S1159F, n = 3; S1159P, n = 8).  To investigate further the action of ivacaftor on S1159F-CFTR, we used excised membrane patches bathed in symmetrical 147 mM Cl^–-rich solutions.  For wild-type CFTR, ivacaftor potentiation of P_o was concentration-dependent, but voltage-independent, while its current-voltage (i-V) relationship was linear and unaffected by increasing concentrations of the drug (n = 5).  Although ivacaftor potentiation of the P_o of S1159F-CFTR was voltage-independent, P_o values were maximal at ivacaftor (100 nM) and not further increased at ivacaftor (1 µM) (n = 3–6).  However, ivacaftor (1 µM) caused voltage-independent inhibition of the i-V relationship of S1159F-CFTR (control, γ = 9.63 ± 0.49 pS; ivacaftor, γ = 5.55 ± 0.97 pS (n = 4); P < 0.001; means ± SD, Student’s paired t-test), revealing that the drug’s action on this CFTR variant is reduced by allosteric channel inhibition.  In conclusion, the molecular basis of CFTR dysfunction caused by the S1159F and S1159P variants is class II (defective processing), III (defective regulation) and IV (defective conduction), necessitating the use of combinations of CFTR modulators to optimal restore their channel activity.  Supported by the NIH, CF Foundation Therapeutics and CF Trust.