In pancreatic beta-cells, cytosolic [ATP (4-)] critically controls insulin secretion via inhibition of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4: 4 stoichiometry of an inwardly rectifying K+ channel subunit (Kir6. 2) plus a regulatory sulfonylurea receptor. To elucidate stoichiometry of ATP (4-) action, we analyzed ATP (4-) sensitivity of channels coassembled from wild-type Kir6. 2 and a loss of ATP (4-) sensitivity mutant (G334D). Concentration-inhibition curves for cDNA ratios of 1: 1 or 1: 10 resembled those for channel block resulting from interaction with 1 of 4 sites, whereas models for inhibition requiring occupation of 2, 3, or 4 sites were incongruous. Random assembly of wild-type Kir6. 2 with the G334D mutant was confirmed by controls, which assessed the effect of an additional mutation that induced strong rectification (N160D). We conclude 4 identical noncooperative ATP (4-) sites to be grouped within 1 KATP channel complex, with occupation of 1 site being sufficient to induce channel closure. This architecture might facilitate coupling of [ATP (4-)] to insulin secretion and may protect against diabetic dysregulation resulting from heterozygous mutations in Kir6. 2.