Abstract: The effect of morphine on serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the CNS of unanesthetized rats was investigated by microdialysis. Morphine was administered either subcutaneously, by local perfusion into the diencephalon, or by intraraphe microinjection. Systemic administration of morphine resulted in a significant increase in both extracellular 5-HT and 5-HIAA in the diencephalon. The effect of morphine on 5-HT was dose dependent during local perfusion of the diencephalon with inhibitors of uptake or monoamine oxidase. Systemic morphine also produced significant increases in extracellular 5-HT in the striatum and hippocampus during uptake inhibition. The site of opioid effects on 5-HT was tested by locally perfusing morphine into the diencephalon. This had no effect on 5-HT or 5-HIAA. In contrast, intraraphe injection of morphine caused a dose-dependent increase in extracellular 5-HT and 5-HIAA in the diencephalon. These results suggest that systemic morphine induces an increase in 5-HT release in widespread areas of the forebrain. This appears to be due to an effect on 5-HT cell bodies and not on 5-HT nerve endings in projection sites.