The effects of infusing N-methyl-d-aspartate (NMDA) into the raphe nuclei on release of 5-HT in this brain region and also the frontal cortex of the same animal were studied using in vivo microdialysis in freely moving rats. Infusion of 25 microM NMDA into the raphe led to a substantial decrease in dialysate 5-HT in this region and a prolonged increase in terminal 5-HT release in the frontal cortex. These effects were blocked by the specific NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (D-AP5; 100 microM). When 25 microM NMDA was co-infused into the raphe with the selective 5-HT1A receptor antagonist (N-¿2-¿4-(2-methoxyphenyl)-1-piperazinyl¿ethyl-N-(2-pyridinyl) cyclohexanecarboxamide) (WAY-100635; 1.0 microM) the effect of NMDA infusion was unaltered. WAY-100635 infused alone into the raphe did not alter local 5-HT or extracellular 5-HT in the cortex. Infusion of 100 microM NMDA into the raphe was followed by an increase in local dialysate 5-HT and a decrease in 5-HT release in the cortex. These changes were reversed by D-AP5. Following infusion of 100 microM NMDA with 1.0 microM WAY-100635 into the raphe local 5-HT release was still increased, however, the decrease in 5-HT observed in the frontal cortex was abolished. These data suggest that the degree of NMDA receptor activation leads to dramatically different outcomes with regard to serotonergic transmission to the frontal cortex. Furthermore, there appears to be a differential role of the 5-HT1A autoreceptor in regulating these effects. These data are discussed in relation to other studies on the regulation of serotonergic transmission in ascending pathways.