The respiratory rhythm is generated by specialized neurons in the medulla oblongata. Glial cells stabilize this rhythm. The neuropeptides substance P (SP), thyrotropin-releasing hormone (TRH) and the biogenic amine serotonin (5-HT) are potent stimulators of the respiratory network. However, the modulation of glial cells, in particular astrocytes, by SP, TRH, and 5-HT is unknown. To study, whether and how these neuromodulators evoke or modulate changes of astroglial calcium signalling, we bulk-loaded acutely isolated brainstem slices of early postnatal mice with the membrane-permeable fluorescent Ca²⁺ -indicator Oregon Green. We used a transgenic mouse line, in which astrocytes express the monomeric red fluorescent protein mRFP1 (TgN (hGFAP-mRFP1)). In addition, astrocytes could be identified by Ca²⁺ influxes in response to application of a low extracellular K+ concentration (0.2 mM). SP and TRH as well as 5-HT and the 5-HT2R agonist a-methyl-hydroxytryptamin (a-me-HT) induced astroglial calcium transients of different kinetics. In the absence of neuronal activity (blocked by 500 nM tetrodotoxin), 5-HT, TRH and a-methyl-HT, but not SP, evoked calcium transients. Since it is well known that any rise in intracellular calcium of astrocytes leads to a vesicular release of glutamate, it is tempting to speculate that TRH and 5-HT constitute a modulatory mechanism of synaptic function in the respiratory network via a direct effect on astrocytes.