The respiratory rhythm is generated by specialized neurons in the medulla oblongata. Astrocytes can stabilize this rhythm. Serotonin is an important modulator of neurotransmission in the respiratory network and dysregulation of serotonin signalling appears to be involved in the pathology of the Rett syndrome. We therefore addressed the potential role of astrocytes in serotonergic signalling of the ventral respiratory group (VRG) by analysing serotonin induced astroglial calcium signalling, using conventional fluorescence microscopy as well multifocal- multiphoton microscopy. Both, the application of serotonin (50 mM) and the specific serotonin receptor 2 agonist a-methyl- hydroxytryptamine (a-me-HT; 50 mM) elicited calcium oscillations in astrocytes of the VRG. In 30.2 ± 16.7 % identified astrocytes, loaded with Oregon green BAPTA-1 AM loaded cells (n=272), showed a-me-HT-induced calcium oscillation also in the absence of neuronal activity (blocked by 500 nM TTX). These data were complimented by immunohistochemical data showing serotonin receptor 2a expression on astrocytes in the ventral respiratory group. Taken together our data suggest that astrocytes are involved in serotonin signalling of the VRG and therefore may indirectly contribute to the pathology of Rett syndrome.