Sustained activation of poly(ADP-ribose) polymerase-1 (PARP-1) and extracellular signal-regulated kinases 1/2 (ERK1/2) both promote neuronal death. Here we identify a direct link between these two cell death pathways. In a rat model of hypoglycemic brain injury, neuronal PARP-1 activation and subsequent neuronal death were blocked by the ERK1/2 inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059). In neuron cultures, PARP-1-mediated neuronal death induced by N-methyl-d-aspartate, peroxynitrite, or DNA alkylation was similarly blocked by ERK1/2 pathway inhibitors. These inhibitors also blocked PARP-1 activation and PARP-1-mediated death in astrocytes. siRNA down-regulation of ERK2 expression in astrocytes also blocked PARP-1 activation and cell death. Direct effects of ERK1/2 on PARP-1 were evaluated by using isolated recombinant enzymes. The activity of recombinant human PARP-1 was reduced by incubation with alkaline phosphatase and restored by incubation with active ERK1 or ERK2. Putative ERK1/2 phosphorylation sites on PARP-1 were identified by mass spectrometry. Using site-directed mutagenesis, these sites were replaced with alanine (S372A and T373A) to block phosphorylation, or with glutamate (S372E and T373E) to mimic constitutive phosphorylation. Transfection of PARP-1 deficient mouse embryonic fibroblasts with the mutant PARP-1 species showed that the S372A and T373A mutations impaired PARP-1 activation, whereas the S372E and T373E mutations increased PARP-1 activity and eliminated the effect of ERK1/2 inhibitors on PARP-1 activation. These results suggest that PARP1 phosphorylation by ERK1/2 is required for maximal PARP-1 activation after DNA damage.