Central CO₂ chemoreceptive neurons in the caudal solitary complex (cSC) are stimulated by hyperoxia via a free radical mechanism. Hyperoxia has been shown to increase superoxide and nitric oxide in the cSC, but it remains unknown how changes in pCO₂ during hyperoxia affect the production of O₂-dependent reactive oxygen and nitrogen species (RONS) downstream that can lead to increased levels of oxidative and nitrosative stress, cellular excitability, and potentially, dysfunction. We used real time fluorescence microscopy in rat brain slices to determine how hyperoxia and hypercapnic acidosis (HA) modulate one another in the production of key RONS, as well as colorimetric assays to measure levels of oxidized and nitrated lipids and proteins. We also examined the effects of CO₂ narcosis and hypoxia prior to euthanasia and brain slice harvesting, as these neurons are CO₂ sensitive and hypothesized to employ CO₂/H⁺ mechanisms that exacerbate RONS production and potentially oxidative stress. Our findings show that hyperoxia ± HA increases the production of peroxynitrite and its derivatives, while increases in Fenton chemistry are most prominent during hyperoxia + HA. Using CO₂ narcosis prior to euthanasia modulates cellular sensitivity to HA post-mortem and enhances the magnitude of the peroxynitrite pathway, but blunts the activity of Fenton chemistry. Overall, hyperoxia and HA do not result in increased production of markers of oxidative and nitrosative stress as expected. We postulate this is due to antioxidant and proteosomal removal of damaged lipids and proteins to maintain cell viability and avoid death during protracted hyperoxia.