Methanesulfonanilide derivatives such as dofetilide are members of the widely used Class III group of cardiac antiarrhythmic drugs. A methanesulfonanilide-sensitive cardiac current has been identified as I-Kr, the rapidly activating component of the repolarizing outward cardiac K+ current, I-K.I-Kr may be encoded by the human ether-related gene (hERG), which belongs to the family of voltage-dependent K+ (K-v) channels having six putative transmembrane segments. The hERG also expresses an inwardly rectifying, methanesulfonanilide-sensitive K+ current. Here we show that hIRK, a member of the two-transmembrane-segment family of inward K+ rectifiers that we have cloned from human heart, is a target for dofetilide. hIRK currents, expressed heterologously in Xenopus oocytes, are blocked by dofetilide at submicromolar concentrations (IC50=533 nmol/L at 40 mV and 20 degrees C). The drug has no significant blocking effect on the human cardiac K, channels hK(v)1.2, hK(v)1.4, hK(v)1.5, or hK(v)2.1. The block is voltage dependent, use dependent, and shortens open times in a manner consistent with open-channel block. While steady state block is strongest at depolarized potentials, recovery from block is very slow even at hyperpolarized potentials (tau=1.17 seconds at -80 mV). Thus, block of hIRK may persist during diastole and might thereby affect cardiac excitability.