Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel with complex regulation. One approach to restore function to CF mutants is the use of small molecules termed CFTR potentiators that interact directly with CFTR to augment channel gating. In previous research (1), we demonstrated that the fluorescein derivative phloxine B (PB) potentiates CFTR channel gating with high affinity and partially rescues the gating defect of the commonest CF mutant F508del. In the present work, we sought to investigate the phloxine B binding site on CFTR. To address our aim, we exploited pharmacological differences between human and murine CFTR (2). Like the inorganic phosphate analogue pyrophosphate (2), PB potentiates robustly the human CFTR Cl- channel, but is without effect on murine CFTR. To begin to investigate the phloxine B binding site, we employed human (h) murine (m) CFTR chimeras. Because the nucleotide-binding domains (NBDs) and regulatory domain (RD) of CFTR control channel gating, we tested the effects of PB on hmCFTR chimeras containing all or part of murine NBD1, RD or NBD2 on a human CFTR backbone. (hmX is a chimera containing human CFTR sequence except for domain X, which is murine CFTR-derived). We recorded CFTR Cl- currents using excised inside-out membrane patches from CHO cells transiently expressing wild-type and chimeric CFTRs. The pipette (external) solution contained 10 mM Cl-, whereas the bath (internal) solution contained 147 mM Cl- at 37 ○C; voltage was -50 mV. PB (0.1 – 5 μM) enhanced human, hmRD (F653-M837), hmNBD1 (N432-K611) and hmNBD1+2 (N432-K611 + I1226-N1419) CFTR Cl- currents, but not those of hmNBD2 (I1226-N1419) (n = 4 – 11). Unexpectly, at PB (5 μM), the drug concentration causing maximum potentiation of human CFTR, potentiation of hmNBD1 CFTR Cl- currents exceeded greatly that of human CFTR (human, 148 ± 4 %; hmNBD1, 264 ± 26 %; means ± SEM; n = 7; P < 0.05; Student’s t-test). To understand why PB potentiates hmNBD1, we studied single channels. PB (5 μM) enhanced greatly hmNBD1-CFTR activity by locking open channels for longer periods than human CFTR (n = 2). To exclude the possibility that PB might interact with the membrane-spanning domains (MSDs) of CFTR to potentiate channel gating, we tested the effects of PB on the CFTR chimera hmTM1-6:TM7-12 that contains MSD1 and MSD2 of murine CFTR. PB (5 μM) potentiated hmTM1-6:TM7-12 by an amount equivalent to that of human CFTR (hmTM1-6:TM7-12, 138 ± 12 %; n = 6; P > 0.05). We interpret our results to suggest that the binding site of PB involves sequences from both NBD1 and NBD2 with NBD2 providing the key structural determinants for PB binding. Future work will refine the structural elements responsible for PB binding to CFTR.