Glycyrrhetinic acid (GA) derivatives have been used to implicate gap junctions in vasorelaxation attributed to endothelium-derived hyperpolarizing factor (EDHF). The aim of this study was to assess whether GA compounds affect endothelial cell hyperpolarization. Membrane potentials were recorded from dye-identified endothelial and smooth muscle cells of guinea pig coronary and rat mesenteric arteries. GA derivatives had varied effects on the resting membrane potential: depolarization, hyperpolarization, or no effect, depending on the artery. 18alpha-GA (50 microM) had a small variable effect on ACh-induced hyperpolarizations in endothelial cells. 18beta-GA (30 microM) and carbenoxolone (100 microM) significantly reduced ACh-induced hyperpolarizations in both endothelial and smooth muscle cells. Smooth muscle action potentials in rat tail arteries were smaller and slower in the presence of 18beta-GA. Nerve-induced excitatory junction potentials were inhibited by 18beta-GA and carbenoxolone, whereas the time course of their decay initially increased and then decreased. In conclusion, the GA compounds had a range of effects. Their inhibition of the EDHF hyperpolarization and relaxation in the smooth muscle may stem from the inhibition of endothelial cell hyperpolarization.