Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels are expressed in thyroid follicular cells where they play a role in transepithelial ion transport (1, 2). These findings together with the rapid, nongenomic effects of thyroid hormone on different ion channels and transporters (3) suggest that thyroid hormone might have direct effects on CFTR. To test this idea, we examined the acute effects of 3,3′,5-triiode-L-thyronine (T3) and 3,5,3′,5′-tetraiodothyronine (T4) on CFTR Cl- channels in excised inside-out membrane patches from C127 cells stably expressing wild-type human CFTR. The external solution contained 10 or 147 mM Cl- and the internal solution 147 mM Cl-, 0.3 mM ATP and 75 nM PKA at 37 oC; voltage was -50 mV. When added to the internal solution facing the inside of CFTR, physiological concentrations or low micromolar concentrations of T3 were without effect on phosphorylated or prephosphorylated CFTR (n = 10). However, higher concentrations of T3 and T4 (≥ 10 μM) inhibited phosphorylated CFTR. Single-channel studies demonstrated that T3 (50 μM) inhibited CFTR Cl- channels by a small decrease of single-channel amplitude (i) (control, i = -0.81 ± 0.03 pA; T3, i = -0.73 ± 0.03 pA; mean ± SEM; n = 6, p < 0.01; Student’s paired t-test) and a great reduction of open probability (Po) (control, Po = 0.37 ± 0.07; T3, Po = 0.09 ± 0.01, n = 6, p < 0.01). Similar inhibitory effects on i and Po were also observed with T4 (50 μM) (n = 4). T3 (50 μM) reduction of Po was largely due to the prolongation of interburst interval (IBI) separating bursts of channel openings (control, IBI = 158 ± 14 ms; T3, IBI = 667 ± 216 ms, n = 3, p < 0.01). In addition, channel block by T3 was voltage independent (n = 5) and was relieved strongly when high concentrations of ATP (5 mM) were present in the internal solution (n = 4). By contrast, when added to the external solution facing the outside of CFTR, T3 (50 μM) was without effect on phosphorylated CFTR (n = 6). We interpret our data to suggest that (i) physiological concentrations of T3 were without direct acute effect on CFTR; (ii) micromolar non-physiological concentrations of T3 inhibit CFTR primarily by an allosteric blocking mechanism (4) and (iii) the inhibitory binding sites of T3 are located on the cytosolic side of CFTR and might share similarity with the ATP binding sites in the nucleotide-binding domain of CFTR.