Several members of the ENaC/degenerin (DEG) family of ion channels can be activated by specific ligands [1]. Interestingly, a small molecule ENaC activator (S3969) has been synthesized and shown to stimulate human but not mouse ENaC in the αβγ-subunit configuration. Moreover, it was shown that the extracellular loop of human βENaC was essential to mediate this stimulatory effect [2]. Our aim was to identify and characterize the S3969 binding site in human βENaC by combining structure-based computer simulations with site-directed mutagenesis and electrophysiological measurements. ENaC was heterologously expressed in Xenopus laevis oocytes. Channel function was assessed by measuring amiloride-sensitive whole-cell currents (ΔI_ami) using the two-electrode voltage clamp technique. A putative S3969 binding site in the channel’s β-subunit was predicted by atomistic modelling, namely docking and molecular dynamics (MD) simulations based on recently published structural information of ENaC (PDB ID 6WTH; [3]). Values are presented as mean±SEM. One-tailed paired t-test was used for statistical analysis. We confirmed that mouse ENaC (α_mβ_mγ_m; n=19) was insensitive to S3969 in concentrations up to 10 µM, whereas S3969 activated human ENaC (α_hβ_hγ_h) by ~2-fold with an EC₅₀ of ~0.3 µM (n=25). Importantly, when a portion of the extracellular loop of mouse βENaC was replaced by the corresponding portion of human β-ENaC (amino acid residues 211-404, α_mβ_m,h(211-404)γ_m) the chimeric channel could be activated by S3969 ~2-fold with an EC₅₀ of ~0.5 µM (n=11). Functional testing of additional mouse-human chimeric βENaC constructs revealed that the β-thumb domain was critically involved in the stimulatory effect of S3969. Furthermore, structural ENaC analysis suggested that the β-thumb domain participated in forming a putative S3969 binding pocket, which was localized at the β-γ-subunit-interface. Computer simulations predicted the key residues within the binding pocket for coordinating S3969. Particularly stable interactions were observed between S3969 and an arginine βR388, a tyrosine βY406 and a phenylalanine βF391 residue. Introducing point mutations at these positions strongly reduced (βR388H, n=15; βR388A, n=12) or nearly abolished (βY406A, n=18; βF391G, n=18) the stimulatory effect of S3969 on ΔI_ami. MD simulations suggested that binding of S3969 to ENaC caused a conformational change of the channel, which weakened the β-γ-intersubunit interactions and increased the distance between the β-thumb and the γ-palm domains. Consistent with these predictions, the stimulatory effect of S3969 on ENaC was abolished when the β-thumb domain was covalently attached to the γ-palm domain following the introduction of two cysteine residues (βR437C – γS298C) to form a disulfide bridge (ΔI_ami=0.79±0.14 µA before and 0.71±0.13 µA after S3969 application, n=15, n.s.). Importantly, reducing the disulfide bond with DTT partially rescued the stimulatory effect of S3969 on ENaC currents (ΔI_ami=0.76±0.09 µA before and 0.98±0.10 µA after S3969 application, n=15, p<0.001). In conclusion, S3969 interacts with a specific binding pocket in the β-subunit of ENaC. This modifies the molecular interaction of the β- and γ-subunits probably causing a conformational change resulting in channel activation. The molecular characterization of the S3969 binding site may help to identify novel endogenous or pharmacological ENaC modulators with possible physiological and therapeutic implications.