Tertiapin (TPN), a small protein toxin originally isolated from honey bee venom, inhibits only certain eukaryotic inward-rectifier K(+) (Kir) channels with high affinity. We found that a short ( approximately 10 residues) sequence in Kir channels, located in the N-terminal part of the linker between the two transmembrane segments, is essential for high-affinity inhibition by TPN and that variability in the region underlies the great variation of TPN affinities among eukaryotic Kir channels. This short variable region is however not present in a bacterial Kir channel (KirBac1.1) or in many other types of prokaryotic and eukaryotic K(+) channels. Thus, the acquisition in evolution of the variable region in eukaryotic Kir channels has created the opportunity to selectively target the numerous types of Kir channel that play important physiological roles. We also show that TPN sensitivity can be readily conferred onto some Kir channels that currently have no known inhibitors by replacing their variable region with that from a TPN-sensitive channel. In heterologous expression systems, such acquired toxin sensitivity will allow currents carried by mutant channels to be readily isolated from interfering background currents. Finally we show that, in the heteromeric GIRK1/4 channels, the GIRK4 and not GIRK1 subunit confers the high affinity for TPN.