The ATP-sensitive K+ (KATP) channels, e.g. in heart and pancreatic beta-cells, open spontaneously in the absence of intracellular ATP (ATPi). Their unitary conductance is approximately 80 pS with 150 mM extracellular K+. These features are shared by the K+ channels composed of various sulfonylurea receptors (SURs) and Kir6.2, whereas SUR/Kir6.1 channels have a smaller conductance (approximately 35 pS) and do not open spontaneously in the absence of ATPi. To identify the structural elements in Kir6.0 subunits which determine these properties, we analyzed the properties of functional K+ channels composed of SUR2A, the cardiac type SUR, and various chimeras of Kir6.1 and Kir6.2 heterologously expressed in HEK (human embryonic kidney) 293T cells. The analyses indicate that the extracellular linker domain between the two putative membrane-spanning regions is responsible for the difference in the single channel conductance between SUR2A/Kir6.1 and SUR2A/Kir6.2 channels. The cytosolic N-terminal domain of Kir6.2 was mandatory for spontaneous channel opening in the absence of ATPi, although a part of C-terminus was also involved. These results implicate specific regions of Kir6.0 in the spontaneous opening and the single channel conductance.