Neuronal signaling, plasticity and pathologies in CA1 hippocampal neurons are all intimately related to the redox environment and, thus, tissue oxygenation. This study tests the hypothesis that hyperoxic superfusate (95%O₂) causes a time-dependent increase in superoxide anion (·O₂^-) production in CA1 neurons in slices, which will decrease as oxygen concentration is decreased. Hippocampal slices (400µm) from weaned rats were incubated with the fluorescent probe dihydroethidium (DHE), which detects intracellular ·O₂^- production. Slices were loaded for 30 min using 10µM DHE and maintained using 1-sided superfusion or continuously loaded using 2.5µM DHE and maintained using 2-sided superfusion (36°C). Continuous loading of DHE and 2-sided superfusion gave the highest temporal resolution measurements of ·O₂^- production, which was estimated by the increase in fluorescence intensity units per minute (FIU/min±SEM) over 4 hr. Superoxide production (2.5µM DHE, 2-sided superfusion) was greatest in 95%O₂ (6.6±0.4 FIU/min) and decreased significantly during co-exposure with antioxidants (100µM melatonin, 25µM MnTMPyP) and lower levels of O₂ (60, 40 and 20%O₂ at 5.3±0.3, 3.3±0.1 and 1.6±0.2 FIU/min, respectively). CA1 cell death after 4 hr (ethidium homodimer-1 staining) was greatest in 95%O₂ and lowest in 40 and 20%O₂. CA1 neurons generated evoked action potentials in 20%O₂ for >4 hr indicating viability at lower levels of oxygenation. We conclude that ·O₂^- production and cell death in CA1 neurons increases in response to increasing oxygen concentration product (=PO₂ x time). Additionally, lower levels of oxygen (20-40%) and antioxidants should be considered to minimize superoxide-induced oxidative stress in brain slices.