The genetic disease cystic fibrosis (CF) is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial Cl- channel regulated by PKA-dependent phosphorylation and cycles of ATP binding and hydrolysis. The most common CF mutation, F508del, causes a temperature-sensitive folding defect that disrupts CFTR processing and channel gating. Previous studies identified the hydrolysable ATP analogue 2′-deoxy ATP (2′-dATP) as a robust potentiator of wild-type (WT)-, G551D- and G1349D-CFTR channel activity (1-2). The current study aimed to investigate the action of 2′-dATP on the F508del-CFTR Cl- channel. We recorded CFTR Cl- channels in excised inside-out membrane patches from C127 and BHK cells stably expressing human CFTR. Cells expressing F508del-CFTR were cultured at 27°C to overcome the mutant’s processing defect. The external solution contained 10 mM Cl- , and the internal solution 147 mM Cl- and 3 mM Mg2+. 1 mM ATP or 2′-dATP and 75 nM PKA were used to activate CFTR at 37°C; holding potential was -50 mV. 2′-dATP increased the open probability (Po) of WT- and F508del-CFTR more strongly than ATP (ATP: WT, Po = 0.48 ± 0.03, F508del, Po = 0.07 ± 0.01; 2′-dATP: WT, Po = 0.72 ± 0.02, F508del, Po = 0.36 ± 0.05; mean ± SEM; n = 4 – 11, p < 0.05; Student’s t-test). Analysis of the concentration-dependence of channel activity suggested that 2′-dATP gates WT-CFTR with higher affinity and greater efficacy than ATP (ATP: Kd = 260 μM, Po(max) = 0.65; 2′-dATP: Kd = 85 μM, Po(max) = 0.71; n = 5 – 14). Burst analysis indicated that 2′-dATP increased the Po of WT- and F508del-CFTR by enhancing the frequency and duration of channel openings; the predominant effect of 2′-dATP was to enhance channel opening frequency (n = 4 – 11). Interestingly, 2′-dATP completely restored the shorter mean burst duration (MBD) of F508del-CFTR to the level of WT-CFTR (ATP: WT, MBD = 155 ± 13 ms, F508del, MBD = 101 ± 10 ms, p < 0.05; 2′-dATP: WT, MBD = 232 ± 23 ms, F508del, MBD = 258 ± 20 ms; p > 0.05; n = 4 – 11). However, 2’d-ATP did not rescue the thermal instability of F508del-CFTR; at 37°C F508del-CFTR Cl- channels ran down in the presence of either ATP or 2′-dATP (n ≥ 5). We interpret our results to suggest that i) 2′-dATP has a higher affinity for CFTR than ATP; ii) 2′-dATP affects both channel openings and closings to augment channel activity; iii) F508del might have distinct effects on channel gating and thermal stability. Taken together, the data suggest that 2′-dATP might be used to develop new CFTR potentiators for CF.