Dual probe microdialysis was employed to characterize dialysate glutamate levels from the substantia nigra pars reticulata of awake freely moving rats, and to test its sensitivity to alterations in striatal neurotransmission including striatal N-methyl-d-aspartic acid (NMDA) receptor stimulation and blockade. Intranigral perfusion with low (0.1 mm) Ca2+ medium (60 min) did not affect nigral glutamate levels, whereas intranigral perfusion with tetrodotoxin (10 μm, 60 min) increased nigral glutamate levels. Perfusion of KCl (100 mm, 10 min) in the dorsolateral striatum transiently stimulated nigral glutamate levels (maximal increase + 60%), whereas intrastriatal perfusion (60 min) with low Ca2+ medium and tetrodotoxin gradually increased nigral glutamate levels. Intrastriatal perfusion with NMDA (0.1–100 μm, 10 min) dose-dependently stimulated glutamate levels in the substantia nigra pars reticulata. The NMDA (1 μm)-induced increase in nigral glutamate release was transient and maximal (+60% within 20 min), whereas that for NMDA (10 μm) had a slow onset but was long lasting (+35% after 60 min). Lower (0.1 μm) and higher (100 μm) NMDA concentrations were ineffective. The effect of intrastriatal NMDA (1 μm) was prevented by coperfusion with MK-801 (1 μm). Intrastriatal MK-801 (10 μm) alone gradually increased glutamate levels up to +50% after 60 min of perfusion. The present results suggest that glutamate levels in the substantia nigra pars reticulata are sensitive to changes in neuronal transmission in the dorsolateral striatum, and that striatal NMDA receptors regulate nigral glutamate release in both a tonic and phasic fashion.