Regulation of serotonin (5-HT) release may be altered during the development of opioid tolerance and dependency. To test this hypothesis, changes in extracellular 5-HT during prolonged administration of morphine were determined by microdialysis in the dorsal raphe nucleus (DRN) of freely behaving rats. Morphine or placebo pellets were implanted s.c. As compared to placebo, morphine pellets induced a sustained, approximately 50% increase in DRN 5-HT and a significant elevation in hot plate latency during the 12-hr period after implantation. One week later DRN 5-HT had returned to control levels, and implanting additional morphine pellets had no effect on 5-HT or hot plate latency. One day after removing the pellets from rats exposed to morphine for 2 wk, acute challenge with morphine (20 mg/kg, s.c.) had a significantly smaller effect on 5-HT in the DRN as compared to the placebo treatment group. Administration of naltrexone to rats implanted with morphine pellets for 2 wk induced signs of withdrawal and a significant decrease in DRN 5-HT. These results suggest that the regulation of 5-HT release is altered during the development of tolerance to morphine. Thus, DRN 5-HT may be one of the factors involved in the changes in physiology and behavioral state during opioid withdrawal.