ATP-sensitive K+ channels (K-ATP channels) are metabolic sensors formed by association of a K+ channel, Kir6, and an ABC protein, SUR, which allosterically regulates channel gating in response to nucleotides and pharmaceutical openers and blockers. How nucleotide binding to SUR translates into modulation of Kir6 gating remains largely unknown. To address this question, we have used a novel conformational K-ATP channel inhibitor, rhodamine 123 (Rho123) which targets the Kir6 subunit in a SUR-dependent manner. Rho123 blocked SUR-less Kir6.2 channels with an affinity of about 1 µM, regardless of the presence of nucleotides, but it had no effect on channels formed by the association of Kir6.2 and the N-terminal transmembrane domain TMD0 of SUR. Rho123 blocked SUR+Kir6.2 channels with the same affinity as Kir6.2 but this effect was antagonized by ATP. Rho123 block protection by ATP was due to direct binding of ATP to SUR and did not entail hydrolysis since it was not mimicked by AMP, did not require Mg2+ and was reduced by mutations in the nucleotide-binding domains of SUR. These results suggest that Rho123 binds at the TMD0-Kir6.2 interface and that binding of ATP to SUR triggers a change in the structure of the contact zone between Kir6.2 and domain TMD0 of SUR that causes masking of the Rho123 site on Kir6.2.