Phospholipase A₂ (PLA₂) activation enhances glutamatergic excitatory synaptic transmission in substantia gelatinosa (SG) neurons which play a pivotal role in regulating nociceptive transmission in the spinal cord. By using melittin as a tool to activate PLA₂, we examined the effect of PLA₂ activation on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at 0 mV in SG neurons of adult rat spinal cord slices by use of the whole-cell patch-clamp technique. Melittin enhanced the frequency and amplitude of GABAergic and glycinergic sIPSCs. The enhancement of GABAergic but not glycinergic transmission was largely depressed by Na⁺ channel blocker tetrodotoxin or glutamate-receptor antagonists (CNQX and/or APV) and also in a Ca²⁺-free Krebs solution. The effects of melittin on glycinergic sIPSC frequency and amplitude were dose-dependent with an effective concentration of about 0.7 µM for half-maximal effect, and were depressed by PLA₂ inhibitor 4-bromophenacyl bromide or aristolochic acid. The melittin-induced enhancement of glycinergic transmission was depressed by lipoxygenase inhibitor nordihydroguaiaretic acid but not cyclooxygenase inhibitor indomethacin. These results indicate that the activation of PLA₂ in the SG enhances GABAergic and glycinergic inhibitory transmission in SG neurons. The former action is mediated by glutamate-receptor activation and neuronal activity increase, possibly the facilitatory effect of PLA₂ activation on excitatory transmission, while the latter action is due to PLA₂ and subsequent lipoxygenase activation and is independent of extracellular Ca²⁺. It is suggested that PLA₂ activation in the SG could enhance not only excitatory but also inhibitory transmission, resulting in the modulation of nociception.