The functional significance of peroxynitrite in the release of [3H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) increased [3H] GABA release in a dose-dependent manner. These increases in [3H]-GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [3H] GABA by the formation of NO. Two types of superoxide scavengers, Cu2+/Zn2+ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [3H]-GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [3H]-GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [3H]-GABA release from the neurons. These results indicate that NO-induced [3H] GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.