The relative importance of GABAergic and glutamatergic afferents in mediating the effects of mu- and kappa-opioids on serotonin (5-HT) efflux in vivo has not been firmly established. Thus, we used microdialysis in the dorsal raphe nucleus (DRN) of freely behaving rats to study the effect of GABA and glutamate receptor antagonists on opioid-induced changes in 5-HT efflux. Infusing the mu-opioid agonist DAMGO (300 microM) increased extracellular 5-HT in the DRN by approximately 70%. During infusion of the GABA(A) receptor blocker bicuculline (100 microM), extracellular 5-HT increased by approximately 250%, and subsequent infusion of DAMGO decreased 5-HT to approximately 70% above the pre-bicuculline baseline. These data are consistent with the hypothesis that mu-opioids disinhibit 5-HT neurons, an effect attenuated by direct inhibition of 5-HT efflux or inhibition of excitatory influences on 5-HT efflux. To further test this hypothesis, glutamate receptor blockers, AP-5 (1 mM) and DNQX (300 microM), were co-infused with DAMGO. The glutamate receptor antagonists prevented decreases in 5-HT elicited by DAMGO in the presence of bicuculline. This indicates that DAMGO inhibits glutamatergic afferents, which partly offsets the disinhibitory influence of mu-opioids on 5-HT efflux. In contrast, the kappa-opioid agonist, U-50,488 (300 microM), decreased 5-HT by approximately 30% in the DRN. Glutamate and GABA receptor antagonists did not block this effect. In conclusion, mu-opioids inhibit GABAergic and glutamatergic afferents, thereby indirectly affecting 5-HT efflux in the DRN. In contrast, kappa-opioids inhibit 5-HT efflux independent of effects on glutamatergic and GABAergic afferents.