The present study aimed to explore mouse brain regions involved in ambulation promoted by l-menthol. Saline or 400 mg/kg l-menthol was subcutaneously administered to mice, and ambulatory activity was measured for 1 hour. The dose of l-menthol significantly promoted mouse ambulation. Immediately after the measurements, the animals were transcardially perfused with fixative and the brains were collected. The brains were subjected to c-Fos immunocytochemistry. The number of c-Fos like immunoreactivity in 24 brain regions was counted. The number of c-Fos like immunoreactivity in each of the brain regions was compared between l-menthol administered mice and saline administered mice. Relationship between the ambulatory activity and the number of c-Fos like immunoreactivity in the brain regions was examined using multiple regression analysis. Furthermore, relationships between the ambulatory activity and the number of c-Fos like immunoreactivity in each of the brain regions were examined using single regression analysis. These analyses indicated brain regions in which the number of c-Fos like immunoreactivity was related to ambulation promoted by l-menthol.