Proteinase-activated receptors (PARs) have a unique activation mechanism in that a proteolytically-exposed N-terminal region acts as a tethered ligand. A potential impact of PAR on sensory processing has not been fully examined yet. Here we report that synthetic peptides with sequences corresponding to PAR ligands enhance glutamatergic excitatory transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. The frequency of spontaneous EPSC was increased by PAR-1 agonist SFLLRN-NH₂ (by 47 % at 1 µM) with small increases by PAR-2 and -4 agonists (SLIGKV-NH₂ and GYPGQV-OH, respectively; at > 3 µM); there was no change in its amplitude or in holding current at -70 mV. The PAR-1 peptide action was inhibited by PAR-1 antagonist YFLLRNP-OH. TFLLR-NH₂, an agonist which is more selective to PAR-1 than SFLLRN-NH₂, dose-dependently increased spontaneous EPSC frequency (EC₅₀ = 0.32 µM). A similar presynaptic effect was produced by PAR-1 activating proteinase thrombin in a manner sensitive to YFLLRNP-OH. The PAR-1 peptide action was resistant to tetrodotoxin and inhibited in Ca²⁺-free solution. Primary-afferent monosynaptically-evoked EPSC amplitudes were unaffected by PAR-1 agonist. These results indicate that PAR-1 activation increases the spontaneous release of L-glutamate onto SG neurons from nerve terminals in a manner dependent on extracellular Ca²⁺. Considering that sensory processing within the SG plays a pivotal role in regulating nociceptive transmission to the spinal dorsal horn, the PAR-1 mediated glutamatergic transmission enhancement could be involved in a positive modulation of nociceptive transmission.