The medial prefrontal cortex (mPFC) is a cortical brain region characterized by complex cognitive functions, such as working memory and decision making. These complex functions are based on a finely tuned interaction between excitatory pyramidal neurons, inhibitory GABAergic interneurons and astrocytes. Astrocytes as well as neurons express a variety of neurotransmitter receptors, the activation of which leads to an increase in intracellular calcium (Ca2+). The aim of this study was to characterize the purinergic Ca2+ signaling and to identify the involved receptors in astrocytes of the mPFC.Confocal Ca2+ imaging of acute brain slices showed that mPFC astrocytes respond to bath application of adenosine with intracellular Ca2+ signals mediated by the A2A receptor. In addition, astrocytes also appear to express A2B receptors, which also induces an increase in cytosolic Ca2+. Application of adenosine 5'-diphosphate (ADP) induced Ca2+ signals in mPFC astrocytes, which could be significantly reduced by inhibition of the P2Y1 receptor. The use of uridine 5'-triphosphate (UTP) as well as uridine 5'-diphosphate (UDP) also detected the expression of P2Y2 and P2Y6 receptors. The A2A receptor was also shown to mediate ADP-induced Ca2+ signals, presumably due to enzymatic hydrolysis of ADP to adenosine. Inhibition of all identified P2Y receptors resulted in a significantly reduced Ca2+ response to application of the non-hydrolysable P2Y agonist ADPbS in astrocytes with a residual signal of approximately 41%. This indicates the involvement of other purinergic receptors stimulated by ADPbS in addition to the P2Y­1, P2Y2 and P2Y6 receptors in Ca2+ signaling in mPFC astrocytes.