This study examined the effects of morphine and fluid percussion traumatic brain injury (TBI) on the activity of cholinergic neurons in specific areas of the rat brain 12 min after injury. Acetylcholine (ACh) turnover, used as an index of cholinergic neuronal activity, was determined using gas chromatography-mass spectrometry. Although morphine administration alone in general did not significantly affect ACh content and turnover in specific brain areas, morphine administered prior to TBI either prevented injury-induced changes in ACh turnover (dorsal pontine tegmentum) or actually reduced the rate constant for ACh utilization (kACh) and the turnover rate of ACh (TRACh) following injury (thalamus, amygdala, cingulate/frontal cortex, and hippocampus). Thus, the protective effects of morphine against enduring behavioral deficits following TBI may involve the inhibition of central cholinergic neurons.