<Purpose> Efficacy of peppermint or its major component menthol on gut function is generally known. Menthol shows its cooling effect via TRPM8, which can be activated by cold stimulation (lower than 25-28°C). Here, we examined the effect of menthol (M) and synthesized menthol derivative (MD, donated by Takasago International Corporation) on smooth muscle contraction induced by acetylcholine (ACh) using isolated intestine of mice, and action mechanism was investigated with relation to TRP channel activity. <Methods> Animal experiments were approved by the animal care and use committee at Chiba Institute of Science and Maebashi Institute of Technology. After euthanasia of mice (ddy, male, over 6 weeks ages) by deep anesthesia, intestines were isolated and divided into 5 different positions, that is, jejunum, upper part of ileum, lower part of ileum, colon, and rectum. After intestinal fragment was set in the Magnus tube kept at 37°C, intestinal motility was monitored by tension meter and recorded on chart recorder. M/MD were dissolved in DMSO and applied against the intestine at 100 μM (MD) or 200 μM (M) as a final concentration. Amplitude of contraction by ACh with DMSO was shown as 100% (control), and those with M or MD were relatively shown. <Results & Discussion> M/MD inhibited ACh-induced intestinal contractions concentration-dependently. MD showed stronger inhibition than M at the same concentration. Inhibitory effects of M/MD were different from intestinal positions, that is, remarkable inhibition could be observed at jejunum and upper part of ileum. At all intestinal positions except jejunum, inhibitory effects of M/MD on ACh-induced contractions became weak at low temperature (23°C). At this temperature, it is thought that TRPM8 has been already activated. So, these results suggested that TRPM8 might involve in inhibitory effects of M/MD and distribution of TRPM8 might be different from intestinal positions. In order to clarify involvement of TRPM8 on inhibitory effects of M/MD, non-specific TRP channel inhibitor ‘ruthenium red’ or non-specific TRP channel antagonist ‘capsazepine’ was used. In spite of inhibitory condition of TRP channel (maybe TRPM8 too) by applying these reagents (final concentration was 10 μM), inhibitory effects of M on the ACh-induced contractions could be still observed. As ruthenium red and capsazepine are not specific against TRPM8, these reagents may action to other existed TRP channels in intestinal tissues and action of M may appear indirectly. It was reported TRPM8 was expressed at mucosa but not at muscle on small intestine (1), and the presence of TRPM8 in the gut has not yet been established clearly (2). These results and reports suggest that M may act at other sites in intestine in addition to TRPM8.