Vesicular glutamate release from astrocytes depends on mobilization of free Ca2+ from the endoplasmic reticulum (ER), and extracellular space to elevate cytosolic Ca2+ (Ca2+cyt). Although mitochondria in neurons, and other secretory cells, have been shown to sequester free Ca2+ and have been implicated in the modulation of Ca 2+-dependent transmitter release, the role of mitochondria in Ca 2+-dependent glutamate release from astrocytes is not known. A pharmacological approach was taken to manipulate Ca2+ accumulation in mitochondria and thereby affect Ca2+cyt of solitary astrocytes in response to mechanical stimuli. Ca2+cyt responses and levels of glutamate release were measured optically in parallel experiments using a fluorescent Ca2+ indicator and an enzyme-linked assay, respectively. It was observed that inhibiting mitochondrial Ca 2+ accumulation is correlated to increased Ca2+cyt and glutamate release, whereas enhancing mitochondrial Ca 2+ accumulation is correlated to decreased Ca2+cyt and glutamate release. These observations suggest that, in addition to the activity of ER and plasma membrane ion channels, mitochondria modulate Ca2+cyt dynamics in astrocytes and play a role in Ca2+-dependent glutamate release from astrocytes. Copyright © 2008 Society for Neuroscience.