Background: There is evidence to suggest that the midbrain periaqueductal grey (PAG) has a role in migraine and the actions of the anti-migraine drug sumatriptan. In the present study we examined the serotonergic modulation of GABAergic and glutamatergic synaptic transmission in rat midbrain PAG slices in vitro. Results: Serotonin (5-hydroxytriptamine, 5-HT, IC50 = 142 nM) and the selective serotonin reuptake inhibitor fluoxetine (30 μM) produced a reduction in the amplitude of GABAA-mediated evoked inhibitory postsynaptic currents (IPSCs) in all PAG neurons which was associated with an increase in the paired-pulse ratio of evoked IPSCs. Real time PCR revealed that 5-HT1A, 5-HT1B, 5-HT1D and 5-HT1F receptor mRNA was present in the PAG. The 5-HT1A, 5-HT1B and 5-HT1D receptor agonists 8-OH-DPAT (3 μM), CP93129 (3 μM) and L694247 (3 μM), but not the 5-HT1F receptor agonist LY344864 (1 – 3 μM) inhibited evoked IPSCs. The 5-HT (1 μM) induced inhibition of evoked IPSCs was abolished by the 5-HT1B antagonist NAS181 (10 μM), but not by the 5-HT1A and 5-HT1D antagonists WAY100135 (3 μM) and BRL15572 (10 μM). Sumatriptan also inhibited evoked IPSCs with an IC50 of 261 nM, and reduced the rate, but not the amplitude of spontaneous miniature IPSCs. The sumatriptan (1 μM) induced inhibition of evoked IPSCs was abolished by NAS181 (10 μM) and BRL15572 (10 μM), together, but not separately. 5-HT (10 μM) and sumatriptan (3 μM) also reduced the amplitude of non-NMDA mediated evoked excitatory postsynaptic currents (EPSCs) in all PAG neurons tested. Conclusion: These results indicate that sumatriptan inhibits GABAergic and glutamatergic synaptic transmission within the PAG via a 5-HT1B/D receptor mediated reduction in the probability of neurotransmitter release from nerve terminals. These actions overlap those of other analgesics, such as opioids, and provide a mechanism by which centrally acting 5-HT1B and 5-HT1D ligands might lead to novel anti-migraine pharmacotherapies.