Blockade of NMDA receptors by intracortical infusion of 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) increases glutamate (GLU) and serotonin (5-HT) release in the medial prefrontal cortex and impairs attentional performance in the 5-choice serial reaction time task. These effects are prevented by the 5-HT(2A) receptor antagonist, (R)-(+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidine methanol (M100907). We explored the roles of endogenous 5-HT and 5-HT(1A) and 5-HT(2C) receptors in the mechanisms by which M100907 suppresses CPP-induced release of cortical GLU and 5-HT using in vivo microdialysis. CPP raised extracellular GLU and 5-HT by about 250% and 170% respectively. The 5-HT synthesis inhibitor, p-chlorophenylalanine (300 mg/kg), prevented M100907 suppressing CPP-induced GLU release. The effect of M100907 on these rises of GLU and 5-HT and attentional performance deficit was mimicked by the 5-HT(2C) receptor agonist, (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine fumarate, (Ro60-0175, 30 microg/kg) while intra-mPFC (SB242084, 6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline, 0.1 microM), a 5-HT(2C) receptor antagonist, prevented the effect of M100907 on extracellular GLU. The 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxenide trihydrochloride (100 microM) abolished the effect of M100907 on the CPP-induced 5-HT release. The data show that blockade of 5-HT(2A) receptors is not sufficient to suppress the CPP-induced rise of extracellular GLU and 5-HT and suggest that M100907 suppresses GLU release induced by CPP by enhancing the action of endogenous 5-HT on 5-HT(2C) receptors.