It is known that the spinal cholinergic system is involved in opioid-induced analgesia. Previous studies have shown that opioids increase spinal acetylcholine (ACh) levels in sheep, and that cholinergic antagonists administered spinally inhibit opioid analgesia in rats. It is therefore possible that the opioid analgesia, at least in part, is mediated by spinally released ACh. To our knowledge there are no reports in the literature showing changes in spinal ACh levels in response to central morphine. Thus, in the current study we investigated the effects of systemically (intraperitoneal) and supraspinally (stereotaxically in the RVM) administered morphine on spinal ACh levels in rodents. Male Sprague-Dawley rats were anesthetized using pentobarbital and laminectomy was performed to expose L4-L6 spinal segments. The dialyzing tip of a microdialysis probe (CMA Microdialysis Inc., MA) was placed in the dorsal horn in L4-L6 region and dialyzed using normal saline at a flow rate of 3 microliters/min. Dialysate was collected on ice and immediately injected into the column of an HPLC system coupled to an ECD (ESA Biosciences Inc., MA) for ACh quantification. The data from the current study shows that systemic administration of morphine (8 mg/kg) significantly increases the release of ACh in the spinal dorsal horn of rats. Preliminary data on central administration of morphine (20 nmoles in 2 microliters) did not show any significant increase in spinal ACh. Our data indicate that opioid receptors in the RVM have limited role in activating spinal cholinergic system, and systemically administered morphine could be acting on spinal opioid receptors causing an increase in ACh levels. Further experiments are underway to study the effects of spinally administered morphine on spinal ACh levels.