The midbrain periaqueductal gray (PAG) is a major site of cannabinoid-mediated analgesia in the central nervous system. In the present study, we examined the actions of cannabinoids on rat PAG neurons in vitro. In brain slices, superfusion of the cannabinoid receptor agonist WIN55,212-2 inhibited electrically evoked inhibitory and excitatory postsynaptic currents in all PAG neurons. The endogenous cannabinoid anandamide inhibited evoked inhibitory postsynaptic currents in the presence of the anandamide transport inhibitor AM404, but not in its absence. The stable anandamide analog R1-methanandamide also inhibited evoked inhibitory postsynaptic currents. WIN55,212-2 reduced the rate of spontaneous miniature inhibitory postsynaptic currents in normal and Ca(2+)-free solutions, but had no effect on their amplitude distributions or kinetics. The WIN55,212-2-induced decrease in miniature inhibitory postsynaptic current rate was concentration dependent (EC(50) = 520 nM). The effects of cannabinoids were reversed by the CB(1) receptor antagonist SR141716. WIN55,212-2 produced no change in membrane current or conductance in PAG neurons in brain slices and had no effect on Ca(2+)-channel currents in acutely isolated PAG neurons. These findings suggest that cannabinoids act via CB(1) receptors to inhibit GABAergic and glutamatergic synaptic transmission in rat PAG, although the efficacy of endogenous cannabinoids is likely to be limited by uptake and breakdown. Like mu-opioids, cannabinoids act to reduce the probability of transmitter release from presynaptic terminals via a Ca(2+)-independent mechanism. In contrast to mu-opioids, cannabinoids have no direct postsynaptic actions on PAG neurons. Thus, cannabinoids and mu-opioids are likely to produce analgesia within PAG in part by different mechanisms.