The chemical structures of the thyroid hormones triiodothyronine (T₃) and thyroxine (T₄) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel. We therefore tested the acute effects of T₃, T₄ and reverse T₃ (rT₃) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T₃, T₄ and rT₃ (all tested at 50 μM) inhibited CFTR in several ways: they reduced strongly CFTR open probability by impeding channel opening; they decreased moderately single-channel current amplitude and they promoted transitions to sub-conductance states. To investigate the mechanism of CFTR inhibition, we studied T₃. T₃ (50 μM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T₃ was weakly voltage-dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T₃ inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to sub-conductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to sub-conductance states. We conclude that T₃ has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.