The Renal Outer Medullary potassium (K+) channel, ROMK or Kir1.1, is expressed almost exclusively in the nephron where it critically regulates systemic sodium, potassium and water balance. Its unique physiological functions and emerging genetic evidence suggest that ROMK may be a viable drug target for a novel class of diuretic. We recently began a drug discovery campaign employing high-throughput screening and medicinal chemistry to develop the first small-molecule inhibitors with which to explore ROMK's therapeutic potential. One inhibitor, termed VU591, inhibits ROMK with submicromolar affinity and is highly selective for ROMK over Kir2.1, Kir2.3, Kir4.1, Kir6.2/SUR1B, Kir 7.1, Slo1/β1 and Kv1.3 K+ channels and more than 65 other potential off targets. Patch clamp analysis, medicinal chemistry and molecular modeling suggest that VU591 blocks the intracellular pore of ROMK by interacting with at least two channel subunits. Using a comparative homology model of ROMK based on the X-ray structure of Kir2.2, all putative solvent-accessible, pore-lining residues that could mediate VU591 interactions were identified. We are currently using scanning mutagenesis and electrophysiology to assess their role in VU591 block of ROMK. These studies should provide novel insights into the molecular structure of the channel and reveal the first glimpse of a selective small-molecule binding site in ROMK.