The function and molecular expression of ATP-sensitive potassium (K_ATP) channels in murine colonic smooth muscle was investigated by intracellular electrical recording from intact muscles, patch-clamp techniques on isolated smooth muscle myocytes, and reverse transcription polymerase chain reaction (RT-PCR) on isolated cells. Lemakalim (1μM) caused hyperpolarization of intact muscles (17.2±3mV). The hyperpolarization was blocked by glibenclamide (1–10μM). Addition of glibenclamide (10μM) alone resulted in membrane depolarization (9.3±1.7mV). Lemakalim induced an outward current of 15±3 pA in isolated myocytes bathed in 5mM external K⁺ solution. Application of lemakalim to cells in symmetrical K⁺ solutions (140/140mM) resulted in a 97±5 pA inward current. Both currents were blocked by glibenclamide (1μM). Pinacidil (1μM) also activated an inwardly rectifying current that was insensitive to 4-aminopyridine and barium. In single-channel studies, lemakalim (1μM) and diazoxide (300μM) increased the open probability of a 27-pS K⁺ channel. Openings of these channels decreased with time after patch excision. Application of ADP (1mM) or ATP (0.1mM) to the inner surface of the patches reactivated channel openings. The conductance and characteristics of the channels activated by lemakalim were consistent with the properties of K_ATP. RT-PCR demonstrated the presence of K_ir 6.2 and SUR2B transcripts in colonic smooth muscle cells; transcripts for K_ir 6.1, SUR1, and SUR2A were not detected. These molecular studies are the first to identify the molecular components of K_ATP in colonic smooth muscle cells. Together with the electrophysiological experiments, we conclude that K_ATP channels are expressed in murine colonic smooth muscle cells and suggest that these channels may be involved in dual regulation of resting membrane potential, excitability, and contractility.