Glutamate kills sensitive neurons through several steps downstream to receptor activation: increased free Ca2+ levels, activation of various enzymes and accumulation of reactive oxygen species (ROS). We have evaluated in a well established model of neuronal cultures the neuroprotective effects of blocking these mechanisms, either singularly or by combining multiple enzyme inhibition and/or ROS scavenging. In vitro cultures of cerebellar granule cells exposed to a toxic concentration of glutamate (100 microM for 15 min in the absence of Mg2+) combined with several pharmacological treatments. Inhibition of nitric oxide synthase (NOS) and phospholipase A2 (PLA2) were effective in decreasing cell death and the combined treatments showed some degree of additivity. By contrast, inhibition of xanthine oxidase (XO) with allopurinol was uneffective. Antioxidants (in particular vitamin e or vitamin E analogs). protected neurons up to more than 50%. A synergistic effect was demonstrated by the combination of vitamin E and C. On the other hand, antioxidants did not increase the protection granted by enzyme inhibitors, suggesting that they act downstream to NOS and PLA2. In conclusion, NOS and PLA2 activated by Ca2+ influx give rise to reactive oxygen species whose deleterious action can be counteracted either by inhibiting these enzymes or by scavenging the excess of free radicals produced by them. Finally, a moderate protection was obtained by blocking protein synthesis with cycloheximide, suggesting a partial contribution of apoptotic mechanisms to the excitotoxic cell death.