Abstract Long term treatment with L-3,4-dihydroxyphenylalanine (l-DOPA) is associated with several motor complications. Clinical improvement of this treatment is therefore needed. Lesions or high frequency stimulation of the hyperactive subthalamic nucleus (STN) in Parkinson's disease (PD), alleviate the motor symptoms and reduce dyskinesia, either directly and/or by allowing the reduction of the l-DOPA dose. N-methyl-d-aspartate (NMDA) receptor antagonists might have similar actions. However, it remains elusive how the neurochemistry changes in the STN after a separate or combined administration of l-DOPA and a NMDA receptor antagonist. By means of in vivo microdialysis, the effect of l-DOPA and/or MK 801, on the extracellular dopamine (DA) and glutamate (GLU) levels was investigated for the first time in the STN of sham and 6-hydroxydopamine-lesioned rats. The l-DOPA-induced DA increase in the STN was significantly higher in DA-depleted rats compared to shams. MK 801 did not influence the l-DOPA-induced DA release in shams. However, MK 801 enhanced the l-DOPA-induced DA release in hemi-parkinson rats. Interestingly, the extracellular STN GLU levels remained unchanged after nigral degeneration. Furthermore, administration of MK 801 alone or combined with l-DOPA did not alter the STN GLU levels in both sham and DA-depleted rats. The present study does not support the hypothesis that DA-ergic degeneration influences the STN GLU levels neither that MK 801 alters the GLU levels in lesioned and non-lesioned rats. However, NMDA receptor antagonists could be used as a beneficial adjuvant treatment for PD by enhancing the therapeutic efficacy of l-DOPA at least in part in the STN.