The fluorescein derivative phloxine B (2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachlorofluorescein) is a potent modulator of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel [1,2]. Nanomolar to low micromolar concentrations of phloxine B potentiate CFTR Cl^– currents, whereas higher concentrations (≥ 10 μM) inhibit CFTR. To understand better channel potentiation by fluorescein derivatives, we studied bengal rose B (2′,4′,5′,7′-tetrabromo-4,5,6,7-tetraiodofluorescein), ethyl eosin (2′,4′,5′,7′-tetrabromoeosin ethyl ester), eosin Y (2′,4′,5′,7′-tetrabromofluorescein), TCF (4,5,6,7-tetrachlorofluorescein), DCF (2′,7′-dichlorofluorescein) and fluorescein using excised inside-out membrane patches from C127 cells expressing wild-type human CFTR. We also employed molecular docking to study the interaction of fluorescein derivatives with CFTR using a head-to-tail dimer model of CFTR’s nucleotide-binding domains (NBDs) [3]. When added to the intracellular solution, with the exception of TCF and fluorescein that lack halogens in the xanthene moiety of the molecule, all other fluorescein derivatives potentiated CFTR Cl^– currents with the rank order of affinity: bengal rose B (K_d, 0.32 μM) > eosin Y (K_d, 0.62 μM) > ethyl eosin (K_d, 1.72 μM) > phloxine B (K_d, 2.78 μM) > DCF (K_d, 36.24 μM). Single-channel studies demonstrated that fluorescein derivatives augment CFTR Cl^– currents by increasing open probability (P_o, P < 0.05, n = 6–15) with phloxine B (1 μM) and eosin Y (1 μM) both prolonging mean burst duration (MBD, P < 0.05) without changing interburst interval (IBI, P > 0.05), and DCF (20 μM) decreasing IBI (P < 0.05) without changing MBD (P > 0.05, n = 4–15). Molecular docking studies suggest that fluorescein derivatives bind at the interface of the NBD dimer primarily by hydrophobic interactions. They also suggest that the putative binding sites involve sequences from both NBD1 and NBD2 and are distinct from the two ATP binding sites. Interestingly, evaluation of the binding free energy implies that fluorescein derivatives bind tighter to NBD1. We conclude that (i) halogens in the xanthene moiety of the molecule have essential roles for CFTR potentiation and different halogens have distinct effects on channel gating; (ii) chlorines in the benzene ring of the molecule enhance potency; (iii) the carboxylic group in the benzene ring plays little role in potentiation, but is a key determinant of CFTR inhibition; and (iv) fluorescein derivatives may potentiate CFTR Cl^– channel by stabilising the formation of the NBD dimer.