This is the first report on the protective effect of heme oxygenase-1 (HO-1) overexpression against oxidative stress-mediated neuronal cell death and demonstration of a decreased production of oxygen free radicals when HO-1 levels are increased. HO-1 is the heat shock/stress cognate of the heat shock protein 32 family of proteins. A known function of these proteins is alpha-meso bridge-specific cleavage of the heme molecule. For the present study, we used cerebellar granular neurons (CGNs) isolated from homozygous transgenic (Tg) mice that overexpress HO-1 under neuron-specific enolase control and nontransgenic (Ntg) littermates. The Tg mouse CGNs were characterized by increased levels of HO-1 mRNA and protein, a lower resting intracellular calcium concentration, and a reduced HO-1 transcriptional response to glutamate-mediated oxidative stress. Compared with the Ntg neurons, when exposed to glutamate (30 microM or 3 mM), the magnitude of cell viability was increased and the number of cells exhibiting membrane permeability and chromatin condensation were significantly decreased in the Tg CGN cultures. The population of neurons surviving glutamate toxicity decreased when HO-1 activity was inhibited by a peptide inhibitor. The neuroprotective effect by HO-1 was extended to H(2)O(2)-induced cell death. The mechanism of protection may involve in part a reduced production of reactive oxygen species upon exposure to glutamate. We suggest that induction of HO-1 by pharmacological means may be a novel approach to amelioration of oxidative insults to neurons.