We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca²⁺ uptake and physiological Ca²⁺ signaling in rat cortical astrocytes. A rise in cytosolic Ca²⁺ ([Ca²⁺]_cyt), resulting from mobilization of ER Ca²⁺ stores was followed by a rise in mitochondrial Ca²⁺ ([Ca²⁺]_m, monitored using rhod-2). Whereas [Ca²⁺]_cyt recovered within ∼1 min, the time to recovery for [Ca²⁺]_m was ∼30 min. Dissipating the mitochondrial membrane potential (Δψ_m, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca²⁺ uptake and slowed the rate of decay of [Ca²⁺]_cyt transients, suggesting that mitochondrial Ca²⁺ uptake plays a significant role in the clearance of physiological [Ca²⁺]_cyt loads in astrocytes. Ca²⁺ signals in these cells initiated either by receptor-mediated ER Ca²⁺ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 ± 11.2 μm/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca²⁺ uptake into mitochondria as the Ca²⁺ wave traveled across the cell. Collapse of Δψ_m to prevent mitochondrial Ca²⁺ uptake significantly increased the rate of propagation of the Ca²⁺ waves by 50%. Taken together, these data suggest that cytosolic Ca²⁺ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca²⁺ signals.