A controlled, periodic exchange of air between lungs and atmosphere requires a neuronal rhythm generated by a network of neurons in the ventral respiratory group (VRG) of the brainstem. Glial cells, e.g. astrocytes, have been shown to be supportive in stabilizing this neuronal activity in the central nervous system during development. In addition, a variety of neuromodulators including serotonin (5-HT), Substance P (SP), and thyrotropin-releasing hormone (TRH) stimulate respiratory neurons directly. If astrocytes in the VRG, like their neuronal neighbors, are also directly stimulated by neuromodulators, they might indirectly affect the respiratory neurons and consequently the respiratory rhythm. In the present study, we provide support for this concept by demonstrating expression of NK1-R, TRH-R, and 5-HT2-R in astrocytes of the VRG with immunohistochemistry. Additionally, we showed that the external application of the neuromodulators 5-HT, SP, and TRH activate calcium transients in VRG astrocytes. Consequently, we postulate that in the VRG of the neonatal mouse, neuromodulation by SP, TRH, and serotonin also involves astrocytic calcium signaling. © 2008 Wiley-Liss, Inc.