Sulfonylurea receptors SUR1 and SUR2 are the regulatory subunits of K(ATP) channels. Their differential affinity for hypoglycemic sulfonylureas provides a basis for the selectivity of these compounds for different K(ATP) channel isoforms. Sulfonylureas have a 100- to 1000-fold greater affinity for SUR1 vs. SUR2. Structure-activity studies suggested a bipartite binding pocket. Chimeric SUR1 approximately SUR2 receptors have shown TMD2, the third bundle of transmembrane helices, to be part of an "A" site that confers SUR1 selectivity for sulfonylureas. The purpose of this study is to determine the position of the "B" site. Previous photoaffinity labeling studies have placed the B site on the amino-terminal third of SUR and colabeled the associated K(IR). In our study, deletion of TMD0, the first bundle of transmembrane helices, did not compromise labeling. Further deletions into the cytoplasmic linker, L0, eliminated binding and labeling. Alanine substitutions in L0 identified a limited number of conserved residues, Y230 and W232, important for affinity labeling. A fragment of K(IR)6.2, missing M2 and the entire carboxyl terminal, assembles with SUR1 and is affinity labeled, while deletion of 10 or more amino-terminal residues compromises labeling. These studies indicate that the B site involves L0 and the K(IR) amino terminus, elements that are critical for control of channel gating.