Peroxynitrite (ONOO-) is a powerful oxidant which is formed from the reaction between nitric oxide (NO) and superoxide anion. It has therefore been proposed to mediate the toxic actions caused by NO. Since ONOO- may be formed in the central nervous system (CNS) in pathological conditions such as brain ischaemia, we decided to investigate whether this molecule induces the release of the endogenous excitatory amino acids glutamate and aspartate from neurones. We selected as biological model acutely dissociated rat cerebellar granule neurones in suspension to allow a direct interaction between ONOO- and target cells. Peroxynitrite caused a concentration-dependent release of aspartate but not of glutamate from dissociated cerebellar granule neurones. Peroxynitrite-induced aspartate release was inhibited by dithiothreitol, tetrodotoxin, and in Na+-deprived solutions and not affected by EGTA or pre-incubation with the cytosolic Ca2+ chelator BAPTA/AM. Peroxynitrite also induced an increase in intracellular Ca2+ concentration which was not affected in the presence of EGTA. These data show that ONOO- causes release of aspartate from cerebellar granule neurones and that this effect might arise from an alteration of Na+ membrane permeability leading subsequently to reversal of a Na+-dependent plasma membrane transporter of this excitatory amino acid. In addition, ONOO- alters Ca2+ homeostasis likely due to Na+ overload. Taken together, these findings may help and elucidate some of the intimate mechanisms of NO-induced neuronal damage in pathological circumstances.