Control of the mesoaccumbens dopamine (DA) pathway by central serotonin(2C) receptors (5-HT(2C)Rs) involves different 5-HT(2C)R populations located within multiple brain areas. Here, using in vivo microdialysis in halothane-anesthetized rats, we assessed the role of medial prefrontal cortex (mPFC) 5-HT(2C)Rs in the control of basal and activated accumbal DA outflow, to identify the modalities of their recruitment and the role of 5-HT(2C)R constitutive activity. Intra-mPFC injection of the 5-HT(2C)R inverse agonist SB 206553 (0.5 microg/0.2 microL), without effect by itself, decreased accumbal DA outflow induced by morphine (2.5-10 mg/kg, s.c.), haloperidol (0.01 mg/kg, s.c.) or GBR 12909 (2.5 mg/kg, i.p.). Conversely, intra-mPFC injection of the 5-HT(2C)R antagonist SB 242084 (0.5 microg/0.2 microL), without effect by itself, decreased the effect of 10 mg/kg morphine, the only drug enhancing basal 5-HT outflow in the mPFC. The inhibitory effect of SB 206553 on 2.5 mg/kg morphine-stimulated DA outflow was suppressed by the concomitant intra-mPFC injection of SB 242084. Finally, changes of basal DA outflow induced by the 5-HT(2C)R agonist Ro 60-0175 (3 mg/kg, i.p.) or SB 206553 (5 mg/kg, i.p.) were unaffected by intra-mPFC injection of SB 242084. These results, showing that 5-HT(2C)R antagonist and inverse agonist behave differently in vivo, demonstrate that mPFC 5-HT(2C)Rs facilitate activated accumbal DA outflow and that 5-HT(2C)R constitutive activity participates in this interaction.