The effect of systemic morphine on extracellular serotonin (5-HT) in the diencephalon of unanesthetized, unrestrained rats was investigated by in vivo microdialysis coupled to high performance liquid chromatography with electrochemical detection. Administration of morphine resulted in a dose dependent increase in extracellular 5-HT. Significant increases were first seen at a dose of 5 mg/kg, and a maximal increase occurred at 10 mg/kg. This increase was blocked by pretreatment with naltrexone, indicating that the effect of morphine on 5-HT was mediated by opiate receptors. Morphine also had a significant effect on extracellular 5-hydroxyindoleacetic acid (5-HIAA), leading to a gradual increase across a range of doses. The effect of morphine on 5-HT was compared to effects of morphine on nociception and catalepsy. Increases in 5-HT and 5-HIAA were first detected at doses that were analgesic but too low to elicit catalepsy. Consistent with many reports that opioids cause an increase in synthesis and turnover of 5-HT in the rat forebrain, the present results provide more direct evidence that 5-HT release is increased after morphine administration.