The integrity of the prefrontal cortex is critical for the expression of working memory. The prefrontal cortex is innervated by dopaminergic afferents from the ventral tegmental area and glutamatergic afferents from the mediodorsal thalamus. To determine the role of dopaminergic and glutamatergic afferents in the regulation of working memory, rats were trained to perform a spatial delayed alternation task in a T-maze. The microinjection of the ionotropic glutamate antagonists 6-cyano-7-nitroquinoxaline-2,3-dione or 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid into the prefrontal cortex impaired working memory. Consistent with a role for glutamate receptor activation, microinjecting the GABA(B) agonist baclofen into the mediodorsal thalamus produced a dose-dependent disruption of working memory. In contrast, inhibition of the mesocortical dopamine projection was without effect on working memory. The blockade of D1 and/or D2 dopamine receptors with SCH-23390 and sulpiride was without effect on working memory. Likewise, the microinjection of baclofen into the ventral tegmental area did not impair working memory. However, stimulating mu-opioid receptors in the ventral tegmental area with [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin produced a dose-dependent impairment of working memory that was reversed by blocking D1 dopamine receptors with SCH-23390 in the prefrontal cortex. These data demonstrate that increased dopamine tone or reduced glutamate tone in the prefrontal cortex disrupts working memory in a spatial delayed alternation task.