The Kir3.1 K+ channel participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2 Å. The selectivity filter is identical to the Streptomyces lividans K+ channel within error of measurement (r.m.s.d.<0.2 Å), suggesting that K+ selectivity requires extreme conservation of three-dimensional structure. Multiple K+ ions reside within the pore and help to explain voltage-dependent Mg2+ and polyamine blockade and strong rectification. Two constrictions, at the inner helix bundle and at the apex of the cytoplasmic pore, may function as gates: in one structure the apex is open and in the other, it is closed. Gating of the apex is mediated by rigid-body movements of the cytoplasmic pore subunits. Phosphatidylinositol 4,5-biphosphate-interacting residues suggest a possible mechanism by which the signaling lipid regulates the cytoplasmic pore.