Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel with characteristic structural similarities to ABC Transporters. Recent cryo-EM studies (Zhang et al., 2018) have revealed a large internal vestibule in the ion permeation pathway of activated CFTR proteins (i.e., phosphorylated, ATP-bound state). We and others lately identified a binding site at the external end of this vestibule for CFTR_inh-172 (Young et al., 2024; Gao et al., 2024), a known voltage-independent CFTR inhibitor. These cryo-EM data also suggest that the binding of CFTR_inh-172 induces conformational changes in CFTR’s two transmembrane segments TM8 and TM12 to obstruct the external end of the pore. Interestingly, our patch-clamp electrophysiological studies show that GlyH-101, another membrane-permeant CFTR blocker, not only blocks CFTR from the external side of the membrane in a voltage-dependent manner (zd = 0.37 ± 0.02; Kd(0) = 3.2 ± 0.8 uM, n = 3) as reported previously (Muanprasat et al., 2004) but also inhibits CFTR currents from the cytoplasmic end with little voltage dependence, suggesting the presence of two distinct binding sites for GlyH-101. Despite structural differences between CFTR_inh-172 and GlyH-101, they are similar in size and polarized charge. We thus hypothesize that GlyH-101 and CFTR_inh-172 may share the same binding pocket—or at least their binding sites may overlap with each other. Indeed, molecular docking shows that GlyH-101 can be accommodated in the same area where CFTR_inh-172 binds. A poorly membrane-permeant GlyH-101 analog (GlyH-101-1) was synthesized by adding a hydrophilic polyethylene glycol tail to the C6 position of the naphthalene ring in GlyH-101. External GlyH-101-1 blocks whole-cell CFTR current in a voltage-dependent manner (zd = 0.30 ± 0.01, Kd(0) = 134 ± 18 uM, n = 5); however, when applied to the cytoplasmic side of an excised inside-out membrane patch, it blocks CFTR in a voltage-independent manner (zd = 0.04 ± 0.001, Kd(0) = 45 ± 17 uM, n = 4). Single-channel data suggest two steps of inhibition: a binding step leading to a short-lived blocked state and a stabler blocked state likely reflecting binding-induced conformational changes. Examining the effects of mutations at the residues involved in the binding of CFTR_inh-172 on the GlyH-101 blockade is underway to further test our hypothesis.