In vivo microdialysis was used to determine if morphine produces increases in extracellular serotonin in specific brain sites. With citalopram included in the dialysis solution to block reuptake, serotonin was measured in 11 brain sites of unanesthetized rats. After systemic morphine (10 mg/kg, s.c.), increases in extracellular serotonin were observed in the nucleus accumbens, amygdala, frontal cortex, striatum, thalamus, hypothalamus and ventral hippocampus. These seven areas are innervated either by dorsal raphe nucleus projections alone, or by projections from both dorsal and median raphe nucleus. In contrast, serotonin was not significantly increased in the medial septal nucleus and dorsal hippocampus. These two areas are selectively innervated by projections from the median raphe nucleus. After systemic morphine, serotonin was increased in the dorsal raphe nucleus, but not in the medial raphe nucleus. Local infusion of morphine through a microdialysis probe in the dorsal raphe nucleus induced a dose-dependent increase of serotonin in the nucleus accumbens, but not in the medial septum. In contrast, infusion of morphine into the median raphe nucleus had little effect on serotonin in either the nucleus accumbens or septum. Infusion of morphine into either the dorsal or median raphe nucleus elicited increased behavioral activity and hyperthermia. These data provide evidence that morphine acts in the area of the dorsal raphe nucleus, but not the median raphe nucleus, to enhance serotonin release in specific forebrain sites, and that the increases in serotonin in the dorsal raphe nucleus projection sites are not an indirect effect of changes in behavioral state or body temperature.