Hydrogen peroxide (H2O2) accumulates during hypoxic-ischemic brain injury and may mediate neurotoxicity in the immature brain. To determine whether H2O2 causes maturation specific neurotoxicity, primary neuronal cultures were exposed to H2O2 (25, 50, 100 microM) for 5 min or 24 h during in vitro development, and toxicity was assessed. Immature neurons incurred marked and dose dependent injury after both brief and prolonged H2O2 exposures, and marked dose dependent death following prolonged H2O2 exposures. Mature neurons incurred marked injury following prolonged but not brief H2O2 exposures, and were relatively resistant to H2O2 induced death following both brief and prolonged exposures. Thus, H2O2 is selectively toxic to immature neurons in vitro. Neuronal vulnerability to H2O2 during in vivo development is unknown and warrants investigation.