Neuronal lesions and peripheral denervation in Chagas' disease are related to local inflammation; however, the pathogenic mechanisms of neuronal lesions in the heart and megavisceras are still unclear. We investigated the involvement of nitric oxide (NO) on neuronal lesion in co-cultures of neurons and macrophages. Trypanosoma cruzi-infected and interferon-gamma (IFN-gamma)-activated co-cultures of neurons and wild-type (WT) macrophages showed significant reduction of both neuronal survival and neurite density. These findings correlated with the levels of NO and the expression of inducible nitric oxide synthase (iNOS). Accordingly, neuronal survival rate in the co-cultures was recovered to control levels by treatment of the cultures with the iNOS inhibitor, aminoguanidine. Moreover, neither neuronal survival nor the neurite density was affected in the co-cultures when the macrophages were harvested from iNOS-deficient mice. These results demonstrate that iNOS-derived NO is the major molecule involved in neuronal damage mechanism in our in vitro model of Chagas' disease neuropathology.