Cyclooxygenase-2 is harmful in models of cerebral ischemia yet plays a protective role in preconditioning-induced ischemic tolerance in the heart. This study examined the mechanisms underlying cyclooxygenase-2-mediated neurotoxicity and preconditioning-induced neuroprotection in an in vitro model of cerebral ischemia. Inhibition of cyclooxygenase-2 protects cortical neuronal cultures from death induced by oxygen–glucose deprivation and reduces oxygen–glucose deprivation-induced increases in intracellular Ca 2+ ([Ca 2+] i ). In the present study, we determined if prostaglandin E 2 (PGE 2) is responsible for this cyclooxygenase-2-mediated effect. Rat cortical cultures expressed mRNA for the prostanoid EP 1–EP 4 receptors. PGE 2 reversed the attenuation in [Ca 2+] i and the protection offered by cyclooxygenase-2 inhibition during oxygen–glucose deprivation. These effects likely occur via activation of the prostanoid EP 1 receptor since blocking this receptor during oxygen–glucose deprivation reduced [Ca 2+] i and neurotoxicity. Next, we considered if the moderate activation of this pathway, by preconditioning cultures with sub-lethal oxygen–glucose deprivation, influenced the development of tolerance to an otherwise lethal oxygen–glucose deprivation insult, 48 h later. Inhibition of cyclooxygenase-2 during oxygen–glucose deprivation–preconditioning abolished preconditioning-induced protection. Furthermore, cultures were rendered tolerant to oxygen–glucose deprivation by the transient exposure to exogenous PGE 2 24 h prior to the insult, indicating that this product of the cyclooxygenase-2 pathway is sufficient to induce ischemic tolerance. This study shows that cyclooxygenase-2 and PGE 2 are involved in both oxygen–glucose deprivation-induced neurotoxicity and preconditioning-induced neuroprotection. While neurotoxic in the context of lethal oxygen–glucose deprivation, the moderate activation of this signalling pathway confers ischemic tolerance.