Twitch tensions of indirectly stimulated diaphragm muscles of mice were blocked by a combination of paeoniflorin (PF) and glycyrrhizin (GLR). The mechanism of this effect was studied electropharmacologically. When twitch responses were completely prevented, miniature end-plate, end-plate, and muscle action potentials were still observed when PF and GLR were combined, suggesting that the mechanism is postsynaptic. Potential amplitudes induced by acetylcholine (ACh), which was injected iontophoretically, were inhibited by about 70% by PF (25 micrograms/ml) plus GLR (75 micrograms/ml), although neither agent alone caused an inhibition. The combined ratio (PF:GLR = 1:3) by concentrations (g/ml) potentiated both the inhibition of ACh potential amplitudes and the depolarization of resting membrane potentials. These results indicate that the effect of combined PF and GLR is to depolarize the muscle membrane and to block ACh-receptor-linked processes. In chemically skinned (saponin-treated) muscles, the tension induced by 0.39 microM of free calcium was inhibited by PF (300 micrograms/ml), but it tended to be increased by GLR (300 micrograms/ml). Caffeine-induced contractures in the skinned muscles was not influenced by PF, and they tended to be decreased with GLR treatment. Thus, in muscles with sustained depolarization, these combined compounds seem to block intracellular Ca2+ movement.