Peroxynitrite (ONOO-) is thought to be involved in the neurodegenerative process. To screen for neuroprotective compounds against ONOO- -induced cell death, we developed 96-well based assay procedures for measuring surviving cell numbers under oxidative stress caused by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1), a generator of ONOO-, and sodium N,N-dietyldithiocarbamate trihydrate (DDC), an inhibitor of Cu/Zn superoxide (O2-) dismutase. Using these procedures, we obtained a microbial metabolite that rescued primary neuronal cells from SIN-1-induced damage, but not from DDC-induced damage. By NMR analysis, the compound was identified as neoechinulin A, an antioxidant compound that suppresses lipid oxidation. We found that the compound rescues neuronal cells such as primary neuronal cells and differentiated PC12 cells from damage induced by extracellular ONOO-. However, non-neuronal cells, undifferentiated PC12 cells and cells of the fibroblast cell line 3Y1 were not rescued. Neoechinulin A has scavenging, neurotrophic factor-like and anti-apoptotic activities. This compound specifically scavenges ONOO-, but not O2- or nitric oxide (NO). Similar to known neuroprotective substances such as nerve growth factor and extracts of Gingko biloba leaves, neoechinulin A inhibits the SIN-1-induced activation of caspase-3-like proteases and increases NADH-dehydrogenase activity. These results suggest that neoechinulin A might be useful for protecting against neuronal cell death in neurodegenerative diseases.