The K(ir) inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (K(ir)1-K(ir)7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective K(ir)2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of K(ir)2.1 function. Here we report one potent K(ir)2.1 inhibitor, ML133, which inhibits K(ir)2.1 with an IC(50) of 1.8 μM at pH 7.4 and 290 nM at pH 8.5 but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on K(ir)1.1 (IC(50) > 300 μM) and displays weak activity for K(ir)4.1 (76 μM) and K(ir)7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the K(ir) family reported to date. Because of the high homology within the K(ir)2 family-the channels share a common design of a pore region flanked by two transmembrane domains-identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent K(ir) inhibitors. Using chimeric channels between K(ir)2.1 and K(ir)1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of K(ir)2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of K(ir)1.1 to those of K(ir)2.1 (N171D and C175I) transplants ML133 inhibition to K(ir)1.1. Together, the combination of a potent, K(ir)2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of K(ir)2 inward rectifier potassium channels.