Isoforms of the mitogen-activated protein kinase ERK have been implicated in both cell survival and cell death. In the present study we explored their role in cell viability in response to oxidative stress. Using the dopaminergic MN9D cell line, we determined that cell death occurred in a concentration-dependent manner after exposure to 6-hydroxydopamine (6-OHDA). The toxicity of 6-OHDA was mediated through generation of reactive oxygen species and was accompanied by a large increase in phosphorylated ERK1/2 but no significant increase in phosphorylated ERK5. 6-OHDA produced a distinct temporal pattern of ERK1/2 activation, with phosphorylated ERK1/2 peaks occurring after 10-15 min (25-fold increase) and 6-24 hr (13-fold increase). Inhibition of the early phosphorylated ERK1/2 peak with U0126 increased the generation of reactive oxygen species by 6-OHDA as well as 6-OHDA-induced toxicity, whereas inhibition of the late peak did not affect 6-OHDA-induced cell death. The time course of phosphorylation of the prosurvival protein CREB mimicked the temporal profile of ERK1/2 activation after 6-OHDA, and blocking the early phospho-ERK1/2 peak also abolished CREB activation. In contrast, activation of caspase-3 by 6-OHDA was delayed, occurring after about 6 hr, and this activation was increased by inhibition of the first phosphorylated ERK1/2 peak. These results suggest that the rapid activation of ERK1/2 in dopaminergic cells by oxidative stress serves as a self-protective response, reducing the content of reactive oxygen species and caspase-3 activity and increasing downstream ERK1/2 substrates.