The contribution of NMDA receptors to regulation of serotonin (5-HT) release was assessed by in vivo microdialysis in freely behaving rats. During infusion of NMDA (30, 100, and 300 microM) into the dorsal raphe nucleus (DRN), 5-HT was increased by approximately 25, 100, and 280%, respectively. Competitive and noncompetitive NMDA-receptor antagonists blocked this effect on DRN 5-HT. Infusion of NMDA (300 microM) into the DRN also produced an 80% increase in extracellular 5-HT in the nucleus accumbens. During infusion of NMDA (100 and 300 microM) into the median raphe nucleus (MRN), 5-HT was increased by approximately 15 and 80%, respectively. NMDA-receptor antagonists blocked this effect on MRN 5-HT. Infusion of NMDA into the MRN also produced a significant increase in hippocampal 5-HT. In contrast, infusion of NMDA into the nucleus accumbens, frontal cortex, or hippocampus produced small decreases in 5-HT in these forebrain sites. Taken together, these results suggest that NMDA receptors in the midbrain raphe, but not the forebrain, can have an excitatory influence on 5-HT neurons and, thus, produce increased 5-HT release in the forebrain. Furthermore, in comparison with the MRN, DRN 5-HT neurons were more sensitive to the excitatory effect of NMDA.