This study evaluated the capacity of mu-opioid and glutamate receptor agonists to differentially regulate the involvement of the GABAergic projection from the ventral pallidum to the mediodorsal thalamus in working memory and locomotor activity. Microinjection of either the ionotropic glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) or the mu agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]enkephalin into the ventral pallidum of male Sprague-Dawley rats produced a dose-dependent impairment in working memory, estimated using a forced delayed alternation task in a T-maze. Performance in a spatial discrimination task without delay was also impaired by glutamate, but not by mu receptor, stimulation. Involvement of the GABAergic projection from the ventral pallidum to the mediodorsal thalamus in mu-opioid-induced impairment of working memory was verified by showing that inhibiting GABA(B) receptors in the mediodorsal thalamus blocked the effect of [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]enkephalin in the ventral pallidum. Similarly, either glutamate or mu-opioid receptor stimulation in the ventral pallidum elicited motor activity, and the motor stimulant effect of the mu agonist was blocked, while that of AMPA is not affected by GABA(B) receptor blockade in the mediodorsal thalamus. Distinction between mu and glutamate receptor stimulation was further revealed by the fact that stimulating mu receptors in the ventral pallidum caused a dose-dependent reduction in extracellular GABA levels, while AMPA was without effect on GABA in the ventral pallidum. These data indicate that stimulating mu-opioid receptors reduces GABAergic tone in the ventral pallidum, which increases activity in the GABAergic projection to the mediodorsal thalamus, thereby impairing working memory. Moreover, it is hypothesized that mu receptors in the ventral pallidum gate the recruitment of working memory into ongoing behavioral activity.