Positron emission tomography (PET) imaging of spinal cord in monkeys with a cholinergic tracer demonstrates increased spinal cholinergic activity in response to an analgesic dose of morphine, and this PET result correlates with measurement of acetylcholine spillover into spinal cord extracellular space induced by morphine, as measured by microdialysis. Previous studies in rats, mice, and sheep demonstrate activation of spinal cholinergic neurons by systemic opioid administration, and participation of this cholinergic activity in opioid-induced analgesia. Testing the relevance of this observation in humans has been limited to measurement of acetylcholine spillover into lumbar cerebrospinal fluid. The purpose of this study was to apply a recently developed method to image spinal cholinergic terminals non-invasively via PET and to test the hypothesis that the tracer utilized would reflect changes in local cholinergic activity. Following Animal Care and Use Committee approval, seven adult male rhesus monkeys were anesthetized on three separate occasions. On two of the occasions PET scans were performed using [(18)F] (+)-4-fluorobenzyltrozamicol ([(18)F]FBT), which selectively binds to the vesicular acetylcholine (ACh) transporter in the presynaptic cholinergic terminals. PET scans were preceded by injection of either saline or an analgesic dose of IV morphine (10 mg/kg). On the third occasion, microdialysis catheters were inserted in the spinal cord dorsal horn and acetylcholine concentrations in dialysates determined before and after IV morphine injection. Morphine increased cholinergic activity in the spinal cord, as determined by blood flow corrected distribution volume of [(18)F]FBT in the cervical cord compared to the cerebellum. Morphine also increased acetylcholine concentrations in microdialysates from the cervical cord dorsal horn. The one animal which did not show increased spinal cholinergic activity by PET from this dose of morphine also did not show increased acetylcholine from this morphine dose in the microdialysis experiment. These data confirm the ability to use PET to image spinal cholinergic terminals in the monkey spinal cord and suggest that acute changes in cholinergic activity can be imaged with this non-invasive technique. Following preclinical screening, PET scanning with [(18)F]FBT may be useful to investigate mechanisms of analgesic action in normal humans and in those with pain.