The effects of pain stimulant and transcutaneous electrical nerve stimulation (TENS) on changes in substance P (SP) levels were investigated in the dorsal root ganglion (DRG) and dorsal horn of the spinal cord of Sprague-Dawley rats. The rats were divided into three groups: control, formalin stimulation and formalin+TENS. In all rats, the right sciatic nerve was attached to a hook electrode for recording, and the right lower leg was connected to a ring electrode. As a pain stimulant for the formalin stimulation group and formalin+TENS group, 50 microliter of a 5%formalin was injected into the right paw. At the same time, TENS (50Hz, 50V) for 5 minutes was applied via the ring electrode to the formalin+TENS group. After completing the stimulation, the components of the sciatic nerve, i. e. the DRG of the fourth to sixth lumbar spinal roots and a part of spinal cord, were removed. SP-like immunoreactivity (SP-LI) in the DRGs was quantified by the ELISA method. For the spinal cord, immunohistochemical staining for SP was carried out using an avidin-biotin immunoperoxidase method. Using an NIH image, the SP grains in the nerve ends, which were seen as dark brown stains on the Rexed laminae I and II in the dorsal horn of the spinal cord, were counted within a 450 micrometer (2) area. The results showed that SP-LI levels of both the DRG and the dorsal horn of the spinal cord in the formalin+TENS group were significantly reduced as compared with formalin stimulation group. Based on the fact that SP is a nociceptive neurotransmitter, the present study suggests that TENS reduces production of SP in the DRG, and shows analgesic effects by suppressing nociception via C-fiber in the peripheral nerves.