We have previously reported that vanilloid compound zingerone, which activates TRPV1 channels in the cell body of primary-afferent neuron, enhances glutamatergic spontaneous excitatory transmission in adult rat spinal lamina II (substantia gelatinosa; SG) neurons by activating TRPA1 channels. Zingerone reversibly and concentration-dependently increased the frequency of spontaneous excitatory postsynaptic current (EPSC; half-maximal effective concentration = 1.3 mM) with a minimal increase in its amplitude. This effect was accompanied by an inward current at -70 mV that was resistant to glutamate-receptor antagonists (a mixture of AMPA-receptor antagonist CNQX and NMDA-receptor antagonist APV). These zingerone effects were repeated and unaffected by voltage-gated Na-channel blocker TTX and selective TRPV1 antagonist capsazepine, whereas being sensitive to non-selective TRP antagonist ruthenium red and TRPA1 antagonist HC-030031. The present study further examined the zingerone activity by applying the blind whole-cell patch-clamp technique to SG neurons of adult rat spinal cord slices. Spontaneous EPSC frequency increase produced by bath-applied zingerone (2 mM) persisted in voltage-gated Ca channel blocker La (30 μM)-, IP₃-induced Ca-release inhibitor 2-aminoethoxydiphenyl borate (200 μM)-containing or nominally Ca-free Krebs solution. On the other hand, the zingerone activity was inhibited by Ca-induced Ca-release inhibitor dantrolene (10 μM). TRPA1 agonist allyl isothiocyanate (100 μM) but not capsaicin (2 μM) inhibited the facilitatory effect of zingerone. With respect to evoked release, zingerone (2 mM) reduced monosynaptic primary-afferent Aδ-fiber and C-fiber EPSC amplitudes. Like allyl isothiocyanate, zingerone (2 mM) increased the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic current recorded at 0 mV in the presence of glycine-receptor antagonist strychnine (1 μM) in a manner sensitive to TTX (0.5 μM). We conclude that the spontaneous L-glutamate release enhancement produced by zingerone is probably due to Ca- but not IP₃-induced Ca-release mechanisms and that zingerone inhibits evoked L-glutamate release while enhancing spontaneous inhibitory transmission in SG neurons.