Immunophilins are receptors for immunosuppressive drugs such as the macrolides cyclosporin A (CsA) and FK506; correspondingly these immunophilins are referred to as cyclophilins and FK506-binding proteins (FKBPs). In particular, CsA targets cyclophilin D (CypD), which can modulate mitochondrial Ca2+ dynamics. Since mitochondria have been implicated in the regulation of astrocytic cytosolic Ca2+ (Cacyt2+) dynamics and consequential Ca2+-dependent exocytotic release of glutamate, we investigated the role of CypD in this process. Cortical astrocytes isolated from CypD deficient mice Ppif -/- displayed reduced mechanically induced Cacyt2+ increases, even though these cells showed augmented exocytotic release of glutamate, when compared to responses obtained from astrocytes isolated from wild-type mice. Furthermore, acute treatment with CsA to inhibit CypD modulation of mitochondrial Ca2+ buffering, or with FK506 to inhibit FKBP12 interaction with inositol-trisphosphate receptor of the endoplasmic reticulum, led to similar reductive effects on astrocytic Cacyt2+ dynamics, but also to an enhanced Ca2+-dependent exocytotic release of glutamate in wild-type astrocytes. These findings point to a possible role of immunophilin signal transduction pathways in astrocytic modulation of neuronal activity at the tripartite synapse. © 2010 Elsevier Ltd.