Introduction: The cystic fibrosis transmembrane conductance regulator (CFTR) channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in various tissues, dysfunction of which leads to diseases such as cystic fibrosis, secretory diarrhoea and kidney disease. In the field of epithelial transport, two drugs assumed to be relatively selective CFTR inhibitors, CFTRinh-172 and GlyH-101 (Ma et al., 2002; Muanprasat et al., 2004), have been staple tools for studying the role of CFTR in vitro. However, the potential off-target effects of these inhibitors on cation channels present in epithelial cells have not previously been addressed. Aims: To investigate the effects of CFTRinh-172 and GlyH-101 on store-operated calcium entry (SOCE) through calcium-permeable channels, as well as on ENaC function. Methods: Calu-3 and HEK293 epithelium-derived cells were loaded with the Ca2+-sensitive fluorescent dye, Fura-2-AM, and SOC channels activated by depleting intracellular Ca2+ stores with 200nM thapsigargin, in Ca2+-free conditions. SOCE was measured by tracking changes in cytosolic Ca2+ using a repeated extracellular Ca2+ addback protocol, alternating between Ca2+-free solution and superfusate containing 1mM Ca2+. Western blot and qPCR were performed to detect CFTR protein and mRNA expression, respectively, in each cell line. Xenopus oocytes were purchased from the European Xenopus Resource Centre and micro-injected with cRNA to express human αβγ-ENaC or δβγ-ENaC. ENaC-expressing oocytes were subjected to two-electrode voltage-clamp (-60 mV holding potential), and changes in transmembrane currents (IM) in response to amiloride (100µM) and CFTR-inhibitors were recorded. All summary data are presented as mean ± SEM, and n denotes the number of independent experiments. Results: Pre-treatment of Calu-3 cells with 20µM CFTRinh-172 irreversibly reduced the SOCE amplitude (24.2±12.3% for 10-min, p=0.0258 and 44.2±10.0%, p<0.0001 for 30-min treatment, n=4-14, Holm-Sidak multiple comparisons vs. vehicle-treated) and rate (52.0±16.4%, p=0.0043 for 30-min treatment, n=4-14, Holm-Sidak multiple comparisons vs. vehicle-treated) in a time-dependent manner (n=4-14, p<0.05, chi-square test). Pre-treatment of HEK293 cells with 20µM CFTRinh-172 or 10µM GlyH-101 also significantly inhibited the amplitude (50.7±10.3%, p=0.0030 and 38.5±8.0%, p=0.0098, respectively; n=5-6, Holm-Sidak multiple comparisons vs. vehicle-treated) and rate (67.5±10.5%, p=0.0015 and 65.7±7.3%, p=0.0015, respectively; n=5-6, Holm-Sidak multiple comparisons vs. vehicle-treated) of SOCE. This was surprising as HEK293 cells expressed negligible levels of CFTR mRNA or protein compared to Calu-3 cells. It was also notable that CFTR inhibitors affected ENaC function. Amiloride-sensitive IM (ΔIami) in Xenopus oocytes expressing αβγ-ENaC were reduced by 20µM CFTRinh-172 (17.5±3.0%, p=0.0004, n=9, Student’s paired t-test) and 10µM GlyH-101 (49.6±4.0%, p=0.0005, n=9, Student’s paired t-test). While GlyH-101 also inhibited ΔIami in δβγ-ENaC expressing oocytes (43.6±2.3%, p<0.0001, n=9, Student’s paired t-test), CFTRinh-172 had a small stimulatory action (8.2±1.1%, p=0.0019, n=9, Student’s paired t-test). Water-injected control oocytes did not respond to amiloride, CFTRinh-172, or GlyH-101. Conclusion: The putative specific CFTR inhibitors, CFTRinh-172 and GlyH-101, exerted CFTR-independent inhibition of SOCE in human epithelial cell lines, and altered ENaC-mediated currents differentially based on subunit composition. Our data indicate that caution is needed when interpreting results using these inhibitors with the intention of dissecting the involvement of CFTR in biological processes.