Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron, and bilirubin. Intense HO-1 immunostaining in the Parkinsonian brain is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP(+)). Seven days after injection of MPP(+) and Ad-HO-1, the brain was isolated for immunostaining and for measurement of dopamine content and inflammatory cytokines. It was found that overexpression of HO-1 significantly increased the survival rate of dopaminergic neurons; reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in substantia nigra; antagonized the reduction of striatal dopamine content induced by MPP(+); and also up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) expression in substantia nigra. Apomorphine-induced rotation after MPP(+) treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by zinc protoporphyrin-IX facilitated the MPP(+)-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 overexpression also protected dopaminergic neurons against MPP(+)-induced neurotoxicity in midbrain neuron-glia cocultures. Overexpression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Pharmacological or genetic approaches targeting HO-1 may represent a promising and novel therapeutic strategy in treating Parkinsonism.