Large subtype-specific differences in the sensitivity of cloned inward-rectifier K⁺ channels of the IRK1, BIR10 and ROMK1 subtype to being blocked by intracellular spermine (SPM) are described. It is shown, by site-directed mutagenesis, that the four orders of magnitude larger SPM sensitivity of BIR10 channels compared to ROMK1 channels may be explained by a difference in a single amino acid in the putative transmembrane segment TMII. This residue, a negatively charged glutamate in BIR10, is homologous to the residue in IRK1 and ROMK1 which has previously been shown to change gating properties and Mg²⁺ sensitivity. Differential block by physiological SPM concentrations is suggested as a major functional difference between subtypes of inward-rectifier K⁺ channels.