High-frequency stimulation of the subthalamic nucleus (STN-HFS) and l-3,4-dihydroxyphenylalanine (l-DOPA) medication are the most used therapeutic approaches in Parkinson's disease (PD), but their beneficial motor effects are burdened by the emergence of cognitive and depressive disorders. Although a reduced serotonergic function has been linked to the psychiatric effects of antiparkinsonian treatments, biochemical evidence supporting this hypothesis is still lacking. By using a microdialysis approach in anesthetized rats, we investigated the ability of STN-HFS (130 Hz, 30 muA, 20 min) and l-DOPA (6-12 mg/kg) to change extracellular levels of serotonin (5-HT) monitored simultaneously in the prefrontal cortex (PFC) and hippocampus (HIPP), two brain regions involved in the regulation of mood and cognition that receive a distinct 5-HT innervation. The results show that STN-HFS inhibited 5-HT levels in the PFC and HIPP of sham-lesioned and 6-hydroxydopamine (6-OHDA)-lesioned rats. The effect elicited by STN-HFS was blocked by the administration of the 5-HT(1A) agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin. l-DOPA (6 and 12 mg/kg) reduced 5-HT levels in the PFC and HIPP of 6-OHDA rats. STN-HFS did not further decrease 5-HT levels induced by l-DOPA, but attenuated l-DOPA-induced dopamine release in the PFC and HIPP. These neurochemical data show that STN-HFS inhibits 5-HT release by modulating serotonergic neuron activity, while the decrease in 5-HT levels induced by l-DOPA may include its direct action inside serotonergic neurons. These results support the premise that antiparkinsonian treatments reduce central serotonergic transmission, which may favor the development of nonmotor side effects in PD.