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Estradiol (E2) modulates the central and peripheral thermoregulatory responses to cold. Menthol is an agonist of transient receptor potential melastatin type 8 (TRPM8), which is a peripheral cold receptor. E2 suppresses menthol-induced elevation of body temperature (Tb) in ovariectomized rats, but the mechanism is unknown. The aim of the present study is to investigate the effect of E2 on uncoupling protein 1 (UCP1), a thermogenic gene, and TRPM8 mRNA levels in ovariectomized rats applied menthol. A silastic tube was implanted in ovariectomized rats with and without E2 underneath the dorsal skin (E2(1) and E2(2) groups), and data loggers for Tb measurement into peritoneal cavity. After application of 10% l-menthol or vehicle to the skin of the whole trunk of rats, Tb was measured for 2 h. The interscapular brown adipose tissue (BAT) and spinal ganglia of cervical, thoracic, and lumbar parts were obtained for RT-qPCR assay. In the menthol application, Tb in the E2(1) group was lower than that in the E2(2) group. The UCP1 mRNA in the BAT, TRPM8 mRNA in the BAT and spinal ganglia in all areas did not differ between the E2(1) and E2(2) groups. In conclusion, the UCP1 and TRPM8 expression in the brown fat did not affect the restriction of the menthol-induced hyperthermia by estradiol in ovariectomized rats.