The periaqueductal gray (PAG) plays a critical role in descending antinociception. In mechanically dissociated rat PAG neurons, pharmacologically separated spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using the nystatin-perforated patch technique. Both DAMGO, a specific mu-opioid receptor agonist, and serotonin inhibited mIPSC frequency in a dose-dependent manner without affecting mIPSC amplitude, respectively, in the same PAG neurons. The presynaptic opioid effect was blocked by a specific mu-opioid receptor antagonist, CTOP. The presynaptic serotonergic effect was mimicked by a specific 5-HT(1A) receptor agonist, 8-OH-DPAT, and blocked by the specific antagonist, NAN-190. These opioidergic and serotonergic inhibitions of GABA release employed the similar intracellular mechanism of opening 4-AP-sensitive K(+) channels via GTP-binding proteins (G-proteins). Subthreshold concentrations of DAMGO (3 nM) significantly decreased mIPSC frequency with subthreshold concentrations of serotonin (3 nM) and this effect was completely blocked by pretreatment with N-ethylmaleimide (NEM), a PTX-sensitive G-protein inhibitor. In contrast, maximum doses of DAMGO (10 microM) did not further inhibit mIPSC frequency with maximum doses of serotonin (10 microM). In conclusion, activation of presynaptic mu-opioid and 5-HT(1A) receptors synergistically inhibited GABA release. These results suggest a cellular mechanism within PAG for the analgesic effectiveness of combined therapies using opioids in conjunction with classes of anti-depressants which increase synaptic serotonin levels.