Prolonged periods of hypoxia are deleterious to higher brain functions and increase the likelihood of developing dementias. Here, we have used fluorimetric techniques to investigate the effects of chronic hypoxia (2.5% O₂, 24h) on Ca²⁺ stores in type I cortical astrocytes, since such stores are crucial to various astrocyte functions, including Ca²⁺ dependent modulation of neuronal activity. Rises of [Ca²⁺]_i evoked by exposure of astrocytes to bradykinin were enhanced following chronic hypoxia, as were transient increases in [Ca²⁺]_i recorded in Ca²⁺-free perfusate. The enhanced responses were due partly to impaired plasmalemal Na⁺ / Ca²⁺ exchange following chronic hypoxia. More importantly, chronic hypoxia increased the Ca²⁺ content of mitochondria (as determined by exposing cells to mitochondrial inhibitors), such that they were unable to act as Ca²⁺ buffers following bradykinin-evoked Ca²⁺ release from the endoplasmic reticulum. Hypoxic enhancement of mitochondrial Ca²⁺ content was also observed in confocal images of cells loaded with the mitochondrial Ca²⁺ indicator, Rhod-2. Confocal imaging of cells loaded with tetramethyl-rhodamine ethyl ester, an indicator of mitochondrial membrane potential, indicated that mitochondria were hyperpolarized in astrocytes following chronic hypoxia. Our findings indicate that hypoxia disturbs Ca²⁺ signaling in type I astrocytes, primarily by causing mitochondrial Ca²⁺ overload.