ATP-sensitive K+(K ATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6. 2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6. 2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6. 2 is highly selective for ATP, and that both the adenine moiety and the β-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6. 2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in K ATP channel gating.