Inward-rectifier K⁺ channels are a group of highly specialized K⁺ channels that accomplish a variety of important biological tasks. Inward-rectifier K⁺ channels differ from voltage-activated K⁺ channels not only functionally but also structurally. Each of the four subunits of the inward-rectifier K⁺ channels has only two instead of six transmembrane segments compared to the voltage-activated K⁺ channels. Thus far, there are no high-affinity ligands that directly target any inward-rectifier K⁺ channel. In the present study, we identified, purified, and synthesized a protein inhibitor of the inward-rectifier K⁺ channels. The inhibitor, called tertiapin, blocks a G-protein-gated channel (GIRK1/4) and the ROMK1 channel with nanomolar affinities, but a closely related channel, IRK1, is insensitive to tertiapin. Mutagenesis studies show that teritapin inhibits the channel by binding to the external end of the ion conduction pore.