Inwardly rectifying potassium (K+) channels (IRKs) maintain the resting membrane potential of cells and permit prolonged depolarization, such as during the cardiac action potential. Inward rectification may result from block of the ion conduction pore by intracellular magnesium (Mg-i(2+)). Two members of this family, IRK1 and ROMK1, which share 40 percent amino acid identity, differ markedly in single-channel K+ conductance and sensitivity to block by Mg-i(2+). The conserved H-5 regions were hypothesized to determine these pore properties because they have this function in voltage-dependent K+ channels and in cyclic nucleotide-gated channels. However, exchange of the H-5 region between IRK1 and ROMK1 had no effect on rectification and little or no effect on K+ conductance. By contrast, exchange of the amino- and carboxyl-terminal regions together transferred Mg2+ blockade and K+ conductance of IRK1 to ROMK1. Exchange of the carboxyl but not the amino terminus had a similar effect. Therefore, the carboxyl terminus appears to have a major role in specifying the pore properties of IRKs.