Transcutaneous electrical nerve stimulation (TENS) is a commonly utilized non-pharmacological treatment for pain. Studies show that low- and high-frequency TENS utilize opioid, serotonin and/or muscarinic receptors in the spinal cord to reduce hyperalgesia induced by joint inflammation in rats. As there is an increase in glutamate and aspartate levels in the spinal cord after joint inflammation, and opioids reduce glutamate and aspartate release, we hypothesized that TENS reduces release of glutamate and aspartate in animals with joint inflammation by activation of opioid receptors. Using microdialysis and HPLC with fluorescence detection, we examined the release pattern of glutamate and aspartate in the dorsal horn in response to either low-frequency (4 Hz) or high-frequency (100 Hz) TENS. We examined the effects of TENS on glutamate and aspartate release in animals with and without joint inflammation. High-frequency, but not low-frequency, TENS significantly reduced spinal glutamate and aspartate in animals with joint inflammation compared with levels in those without joint inflammation. The reduced release of glutamate and aspartate by high-frequency TENS was prevented by spinal blockade of delta-opioid receptors with naltrindole. Thus, we conclude that high-frequency TENS activates delta-opioid receptors consequently reducing the increased release of glutamate and aspartate in the spinal cord.