In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICC) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent non-specific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch clamp technique and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, -GA (10 µM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of lucifer yellow, increased input resistance and decreased cell capacitance in ICC networks suggesting that GA uncoupled ICC's. In patch clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca²⁺ current in a dose-dependent manner without affecting the voltage-dependence of this current. The IC₅₀ for Ca²⁺ currents was 1.9µM which is lower than the concentrations used to block gap junctions. GA also significantly increased large conductance Ca²⁺-activated K⁺ currents but decreased net delayed rectifier K⁺ currents, including 4-AP and TEA-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca²⁺ currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICC's and smooth muscles and inhibition of K⁺ conductances in smooth muscle cells.