In the present study, we investigated the effect of morphine on the extracellular levels of glutamate in the anterior cingulate cortex (ACC) in freely moving rats using in vivo microdialysis coupled to high performance liquid chromatography and electrochemical detection. The results showed that either acute or chronic morphine treatment decreased the extracellular levels of glutamate in the ACC. Naloxone could reverse the decrease induced by chronic morphine treatment. The present study provided the first neurochemical evidence that morphine decreased extracellular levels of glutamate in the ACC, suggesting that glutamate in ACC is involved in the central actions of morphine.