An investigation of the mechanism of peppermint oil action was performed using isolated pharmacological preparations from guinea pig large intestine and patch clamp electrophysiology techniques on rabbit jejunum. Peppermint oil relaxed carbachol-contracted guinea pig taenia coli (IC50, 22.1 micrograms/mL) and inhibited spontaneous activity in the guinea pig colon (IC50, 25.9 micrograms/mL) and rabbit jejunum (IC50, 15.2 micrograms/mL). Peppermint oil markedly attenuated contractile responses in the guinea pig taenia coli to acetylcholine, histamine, 5-hydroxytryptamine, and substance P. Peppermint oil reduced contractions evoked by potassium depolarization and calcium contractions evoked in depolarizing Krebs solutions in taenia coli. Potential-dependent calcium currents recorded using the whole cell clamp configuration in rabbit jejunum smooth muscle cells were inhibited by peppermint oil in a concentration-dependent manner. Peppermint oil both reduced peak current amplitude and increased the rate of current decay. The effect of peppermint oil resembled that of the dihydropyridine calcium antagonists. It is concluded that peppermint oil relaxes gastrointestinal smooth muscle by reducing calcium influx.