Increasing evidence indicates that nitric oxide acts as an intercellular signal transduction molecule in the nervous system. In particular, in vitro studies have demonstrated that nitric oxide is produced in the cerebellar cortex and is responsible for the increases in cyclic GMP seen in response to glutamate receptor activation. In this study, we have combined the technique of intracerebellar microdialysis with a sensitive assay for nitric oxide oxidation products nitrate and nitrite, to assess nitric oxide release directly in awake, freely moving animals. We have found that infusion of N-methyl-D-aspartate via the microdialysis probe results in a dose-dependent increase in cerebellar nitric oxide release. This increase was prevented by prior administration of an N-methyl-D-aspartate receptor antagonist, or the nitric oxide synthase inhibitor NG-nitroarginine. Both these pretreatments also reduced the basal extracellular nitrite and nitrate levels, suggesting that there is a tonic glutamate-induced nitric oxide production in the cerebellum of awake, freely moving animals. These results provide direct evidence for nitric oxide release in response to N-methyl-D-aspartate receptor activation in the adult cerebellar cortex, in vivo. This new approach, coupling microdialysis with the azo dye detection method of Griess, should thus prove useful for the in vivo study of nitric oxide release from various brain regions in response to pharmacological, physiological or behavioral manipulations.