Despite the crucial role that prostaglandins (PGs) have in the sensitization of the central nervous system to pain, their cellular and molecular targets leading to increased pain perception have remained elusive. Here we investigated the effects of PGE₂ on fast synaptic transmission onto neurons in the rat spinal cord dorsal horn, the first site of synaptic integration in the pain pathway. We identified the inhibitory (strychnine-sensitive) glycine receptor as a specific target of PGE₂. PGE₂, but not PGF_2α, PGD₂ or PGI₂, reduced inhibitory glycinergic synaptic transmission in low nanomolar concentrations, whereas GABA_A, AMPA and NMDA receptor-mediated transmission remained unaffected. Inhibition of glycine receptors occurred via a postsynaptic mechanism involving the activation of EP2 receptors, cholera-toxin-sensitive G-proteins and cAMP-dependent protein kinase. Via this mechanism, PGE₂ may facilitate the transmission of nociceptive input through the spinal cord dorsal horn to higher brain areas where pain becomes conscious.