Removal of arginine-framed trafficking motifs (AFTs) allows misfolded membrane proteins to escape the endoplasmic reticulum (ER) quality control, rescuing their cell surface expression [1,2]. The simultaneous replacement of arginine by lysine at positions 29, 516, 555 and 766 (4RK) restored the processing and function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing ΔF508, the most common CF mutation [2]. Another ΔF508 revertant mutation (G550E), located within the conserved LSGGQ motif of nucleotide-binding domain 1 (NBD1) produced, by itself, a similar effect [1]. However, it is unknown whether G550E and 4RK rescue other CF mutants or affect the function of wild-type (WT)-CFTR. Here, we tested the effects of these revertants on WT-, ΔF508- and V562I-CFTR using excised inside-out membrane patches from BHK cells expressing each CFTR variant. The pipette (extracellular) solution contained 10 mM Cl^–, and the bath (intracellular) solution contained 147 mM Cl^–, ATP (1 mM) and PKA (75 nM) at 37°C; voltage was –50 mV. Data are means ± S.E.M. of n observations and statistical analyses were performed using Student’s paired t test. Temperature-rescued ΔF508-CFTR had a reduced open probability (P_o) with dramatically prolonged closures between bursts of openings. In contrast, V562I generated a regulated Cl^– channel with P_o, mean burst duration (MBD) and interburst interval (IBI) similar to WT (WT, P_o = 0.47±0.04, n = 6; ΔF508, P_o = 0.06±0.01, n = 10, p < 0.001; V562I, P_o = 0.45±0.01, n = 5, p > 0.05). 4RK only slightly increased the P_o of WT by doubling MBD and slightly prolonging IBI (4RK, P_o = 0.54±0.04, n = 2). However, G550E almost doubled the P_o of WT by increasing the MBD 4-fold, without affecting IBI (G550E, P_o = 0.77±0.02, n = 3, p < 0.001). 4RK in cis with ΔF508 increased P_o, but it was still lower than WT (ΔF508-4RK, P_o = 0.23±0.02, n = 13, p < 0.001); MBD was enhanced 5-fold, but IBI was unchanged. ΔF508-G550E had a much higher P_o than ΔF508 because MBD was increased 8-fold and IBI decreased 2.5-fold. For V562I, 4RK caused a slight increase in P_o (V562I-4RK, P_o = 0.55±0.07; n = 4; p > 0.05), but G550E greatly enhanced it (V562I-G550E, P_o = 0.80±0.03; n = 8; p < 0.001). Neither 4RK nor G550E altered the IBI of V562I. However, 4RK prolonged MBD of V562I 2-fold and G550E 4-fold. Our data suggest that G550E enhances more strongly than 4RK the gating behaviour of WT, ΔF508 and V462I. They also suggest that V562I might not be a CF-causing mutation. Consistent with this idea, two individuals with the genotype ΔF508/V562I have no clinical signs of CF (P. Pacheco and C. Barreto, personal communications).