Identification of selective ion channel inhibitors represents a critical step for understanding the physiological role that these proteins play in native systems. In particular, voltage-gated potassium (K(V)2) channels are widely expressed in tissues such as central nervous system, pancreas, and smooth muscle, but their particular contributions to cell function are not well understood. Although potent and selective peptide inhibitors of K(V)2 channels have been characterized, selective small molecule K(V)2 inhibitors have not been reported. For this purpose, high-throughput automated electrophysiology (IonWorks Quattro; Molecular Devices, Sunnyvale, CA) was used to screen a 200,000-compound mixture (10 compounds per sample) library for inhibitors of K(V)2.1 channels. After deconvolution of 190 active samples, two compounds (A1 and B1) were identified that potently inhibit K(V)2.1 and the other member of the K(V)2 family, K(V)2.2 (IC(50), 0.1-0.2 μM), and that possess good selectivity over K(V)1.2 (IC(50) >10 μM). Modeling studies suggest that these compounds possess a similar three-dimensional conformation. Compounds A1 and B1 are >10-fold selective over Na(V) channels and other K(V) channels and display weak activity (5-9 μM) on Ca(V) channels. The biological activity of compound A1 on native K(V)2 channels was confirmed in electrophysiological recordings of rat insulinoma cells, which are known to express K(V)2 channels. Medicinal chemistry efforts revealed a defined structure-activity relationship and led to the identification of two compounds (RY785 and RY796) without significant Ca(V) channel activity. Taken together, these newly identified channel inhibitors represent important tools for the study of K(V)2 channels in biological systems.