Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO₂, arterial O₂, and brain activity and is largely constant in the awake state. Although small changes in arterial CO₂are particularly potent to change CBF (1 mmHg variation in arterial CO₂changes CBF by 3%–4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E₂(PgE₂) plays a key role for cerebrovascular CO₂reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices within vivowork in rats and C57BL/6J mice to examine the hemodynamic responses to CO₂and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE₂synthesis. We demonstrate that hypercapnia (increased CO₂) evokes an increase in astrocyte [Ca²⁺]_iand stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE₂(microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE₂is decreased and vasodilation triggered by increased astrocyte [Ca²⁺]_iin vitroand by hypercapniain vivois inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO₂. Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage.SIGNIFICANCE STATEMENTNeuronal activity leads to the generation of CO₂, which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE₂. We demonstrate that hypercapnia (increased CO₂) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE₂production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE₂is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO₂-evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage.