The present study investigated whether 5-HT2C receptors in the ventrotegmental area and prefrontal cortex regulate basal and stimulus-evoked dopamine release in the prefrontal cortex. Using the in vivo microdialysis technique in conscious rats, we studied the effect of a selective 5-HT2C receptor agonist, Ro60–0175, on basal and immobilization stress-induced dopamine release in the prefrontal cortex. Ro60–0175 intraperitoneally (2.5 mg/kg) and into the ventrotegmental area (10 µg/0.5 µL) completely antagonized the effect of stress on extracellular dopamine without altering basal levels. Infusion of 10 µm Ro60–0175 through the cortical probe had no significant effect on basal and stress-induced dopamine release. SB242084 (10 mg/kg), a selective antagonist of 5-HT2C receptors, significantly increased basal extracellular dopamine and completely prevented the effect of intraperitoneal and intraventrotegmental Ro60–0175 on the stress-induced rise of extracellular dopamine, but had no effect itself in stressed rats. The results show that Ro60–0175 suppresses cortical dopamine release induced by immobilization stress through the stimulation of 5-HT2C receptors in the ventrotegmental area. While confirming that endogenous 5-HT acting on 5-HT2C receptors tonically inhibit basal dopamine release in the prefrontal cortex, the present findings suggest that the stimulation of 5-HT2C receptors with an exogenous agonist preferentially inhibit stimulated release.