Activated microglia contribute to cell death in ischemic and neurodegenerative disorders of the CNS. Microglial activation is regulated in part by NF-κB, and the nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1) enhances NF-κB binding to DNA. In this study, the role of PARP-1 in microglia-mediated neurotoxicity was assessed using microglia from wild-type (wt) and PARP-1−/− mice. Cultured microglia were incubated with TNF-α, a cytokine that is up-regulated in many neurological disorders. When stimulated with TNF-α, wt microglia proliferated, underwent morphological changes characteristic of activation, and killed neurons placed in coculture. The effects of TNF-α were markedly attenuated both in PARP-1−/− microglia and in wt microglia treated with the PARP enzymatic inhibitor 3, 4-dihydro-5-[4-(1-piperidinyl) butoxy]-1 (2h)-isoquinolinone. These effects were also blocked by (E)-3-(4-methylphenylsulfonyl)-2-propenenenitrile, which inhibits translocation of NF-κB to the nucleus. TNF-α also up-regulated microglial release of matrix metalloproteinase-9 (MMP-9), an enzyme with potential neurotoxic properties that is transcriptionally regulated by NF-κB. This up-regulation was blocked in PARP-1−/− microglia and in wt microglia by the PARP inhibitor 3, 4-dihydro-5-[4-(1-piperidinyl) butoxy]-1 (2h)-isoquinolinone. Microglia from MMP-9−/− mice were used to evaluate the contribution of MMP-9 to microglial neurotoxicity. MMP-9−/− microglia treated with TNF-α showed substantially reduced neurotoxicity relative to the wt microglia. TNF-α-stimulated wt microglia treated with the MMP inhibitor ilomastat also showed reduced neurotoxicity. These findings suggest that PARP-1 activation is required for both TNF-α-induced microglial activation and the neurotoxicity resulting from TNF-α-induced MMP-9 release.