The effect of morphine on serotonin (5-HT) was examined by microdialysis in unanesthetized and anesthetized rats. In unanesthetized rats, morphine (10 mg/kg, s.c.) produced increases in extracellular 5-HT in nucleus accumbens (n. accumbens) and dorsal raphe nucleus (DRN), but not in the dorsal hippocampus. Similarly, extracellular 5-HT in the n. accumbens, but not the dorsal hippocampus, was increased after morphine (1 mM) was infused for 60 min by reverse dialysis into the DRN. Chloral hydrate, pentobarbital, and ketamine anesthesia had different effects on 5-HT in the n. accumbens. Chloral hydrate induced a transient increase and ketamine a sustained increase in extracellular 5-HT. Pentobarbital caused a sustained decrease. The effects of systemic and intraraphe administration of morphine were abolished by all three anesthetics. Infusion of muscimol, a GABAA receptor agonist, into the DRN also induced a decrease in 5-HT and abolished the effects of morphine on 5-HT in the DRN and n. accumbens. These results are consistent with other evidence suggesting that morphine-induced increases in monoamine neurotransmission are a disinhibitory effect resulting from opioid-mediated inhibition of GABA release. More conclusively, it is apparent that anesthetized animals are inappropriate for testing the effect of morphine on 5-HT neurotransmission.