INWARDLY rectifying K+ channels (IRKs) conduct current preferentially in the inward direction. This inward rectification has two components: voltage-dependent blockade by intracellular Mg2+ (Mg-i(2+))(1-7) and intrinsic gating(8,9). Two members of this channel family, IRK1 (ref. 10) and ROMK1 (ref. 11), differ markedly in affinity for Mg-i(2+) (ref. 12). We found that IRK1 and ROMK1 differ in voltage-dependent gating and searched for the gating structure by large-scale and site-directed mutagenesis. We found that a single amino-acid change within the putative transmembrane domain M2, aspartate (D) in IRK1 to the corresponding asparagine (N) in ROMK1, controls the gating phenotype. Mutation D172N in IRK1 produced ROMK1-like gating whereas the reverse mutation in ROMK1-N171D-produced IRK1-like gating. Thus, a single negatively charged residue seems to be a crucial determinant of gating.