The role of oxygen-derived free radicals, superoxide in particular, in the pathogenesis of neuronal cell death induced by glutamate was studied using primary culture cortical neurons from transgenic mice overexpressing human CuZn-superoxide dismutase. Primary cortical neuron cultures were developed form 15-day-old fetuses of both transgenic mice and their normal littermates. Both human CuZn-superoxide dismutase and host mouse CuZn-superoxide dismutase activities in cultured neurons were identified by native gel electrophoresis followed by nitroblue tetrazolium staining. Cultured neurons grown for 10-12 days in vitro were exposed briefly to 0.5 mM glutamate for 5 minutes, followed by biochemical and morphological examinations at 2 and 4 hours. Our data have demonstrated that glutamate neurotoxicity is significantly reduced in transgenic neurons at 2 and 4 hours following exposure to glutamate, as measured by the intracellular 3-0-methyl glucose space, the efflux of lactate dehydrogenase, and by phase-contrast and bright-field trypan blue staining. These data indicate that transgenic neurons containing two- to threefold the normal amount of CuZn-superoxide dismutase activity are protected against glutamate neurotoxicity in vitro. Our results suggest that oxidative stress play an important role in glutamate-induced neuronal swelling and toxicity.