We have previously reported dual effects of mu-opioids on N-methyl-D-aspartate (NMDA)-receptor-mediated synaptic events in the hippocampal dentate gyrus: an indirect facilitating effect via suppression of GABAergic interneurons (disinhibition) and a direct inhibitory effect in the presence of gamma-aminobutyric acid-A (GABA(A)) antagonists. The cellular mechanism underlying the inhibitory effect of mu-opioids remains to be determined. In the present study we examine the role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in mu-opioid-induced inhibition of NMDA currents in rat hippocampal slices. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were evoked by stimulating the lateral perforant path and were recorded from dentate granule cells with the use of whole cell voltage-clamp techniques in the presence of the GABA(A) antagonist and a non-NMDA type of glutamate receptor antagonist. Two selective mu-agonists, [N-MePhe3, D-Pro4]-morphiceptin and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin, induced dose-dependent inhibition of NMDA EPSCs in a concentration range of 0.3-10 microM. This inhibitory effect could be completely reversed by the opioid antagonists naloxone or prevented by a selective mu-antagonist cyprodime, but was not affected by removal of Mg2+ from the external perfusion medium. Intracellular application of pertussis toxin (PTX) into the granule cell via whole cell recording pipettes completely prevented mu-opioid-induced reduction in NMDA currents, suggesting that a postsynaptic mechanism involving PTX-sensitive G proteins might be responsible for the inhibitory action of mu-opioids. Further studies were conducted to identify the intracellular messengers that coupled with G proteins and transduced the effect of mu-opioids in granule cells. The adenylate cyclase activator forskolin was found to enhance NMDA-receptor-mediated synaptic responses and to reverse the inhibitory effect of mu-opioids. Sp-cAMPS, a specific PKA activator, also enhanced NMDA EPSCs, whereas the PKA inhibitor Rp-cAMPS reduced NMDA EPSCs and occluded further inhibition of the current by mu-opioids. These findings strongly suggest that NMDA receptor function is subject to the modulation by PKA, and that mu-opioids can inhibit NMDA currents through suppression of the cAMP cascade in the postsynaptic neuron. Combined with our previous findings, the present results also indicate that mu-opioids can modulate NMDA-receptor-mediated synaptic activity in a complex manner. The net effect of mu-opioids in the dentate gyrus may depend on the interplay between its disinhibitory action, which facilitates NMDA-receptor-mediated responses, and its inhibitory action on the cAMP cascade.