An inwardly rectifying K(+) conductance closely resembling the human ether-a-go-go-related gene (HERG) current was identified in single smooth muscle cells of opossum esophageal circular muscle. When cells were voltage clamped at 0 mV, in isotonic K(+) solution (140 mM), step hyperpolarizations to -120 mV in 10-mV increments resulted in large inward currents that activated rapidly and then declined slowly (inactivated) during the test pulse in a time- and voltage- dependent fashion. The HERG K(+) channel blockers E-4031 (1 microM), cisapride (1 microM), and La(3+) (100 microM) strongly inhibited these currents as did millimolar concentrations of Ba(2+). Immunoflourescence staining with anti-HERG antibody in single cells resulted in punctate staining at the sarcolemma. At membrane potentials near the resting membrane potential (-50 to -70 mV), this K(+) conductance did not inactivate completely. In conventional microelectrode recordings, both E-4031 and cisapride depolarized tissue strips by 10 mV and also induced phasic contractions. In combination, these results provide direct experimental evidence for expression of HERG-like K(+) currents in gastrointestinal smooth muscle cells and suggest that HERG plays an important role in modulating the resting membrane potential.