Astrocytes, the most abundant cell type in the CNS, are accepted to play essential roles in brain function by supporting neuronal viability and vascular integrity. Whilst astrocytes are not electrically active, their properties are subject to regulation via dynamic changes in intracellular Ca2+ signalling, events that are believed to play a critical role in coordination of astrocyte communication and gliotransmission. GABAB receptors (GABABRs) are heterodimeric G-protein coupled receptors, which mediate slow synaptic inhibition in the brain. Emerging evidence suggests astrocytes express GABABRs, however the role GABABRs play in regulating astrocyte activity remains largely speculative. To determine whether astrocytic GABABRs modulate Ca2+ signalling, cultures were loaded with Fluo-4/AM and dynamic changes in cytosolic Ca2+ levels were analysed using time-lapse confocal microscopy (Terunuma et al., 2015). Exposure of astrocytes to baclofen did not lead to any significant changes in intracellular Ca2+ levels. In contrast, subsequent exposure of astrocytes to the P2 purinoceptor (P2R) agonist ATP rapidly increased intracellular Ca2+ levels, consistent with published studies (Fischer et al., 2009). Importantly, after exposure to ATP, evidence of small baclofen induced Ca2+ transients was observed. To assess whether GABABR signalling is facilitated by pre-exposure to ATP, we used the GABABR antagonist CGP54626. Pre-treatment of astrocytes with CGP54626 abolished baclofen-evoked Ca2+ transients, however, CGP54626 alone did not have any effect on ATP-dependent increases in Ca2+. Collectively, these studies strongly suggest the ability of GABABRs to modulate astrocytic Ca2+ signalling is facilitated by ATP. To analyse the mechanisms by which ATP regulates GABABR activity, we examined its effects on the phosphorylation of S783 and S892 within the GABABR2 subunit, accepted substrates of AMPK and PKA , respectively (Couve et al., 2002; Terunuma et al., 2010). To do so, we used phospho-specific antibodies against these residues, and the ratio of p-S783/R2 and p-S892/R2 was then compared between treatments. Exposure of astrocytes to ATP stimulation significantly increased S783 and S892 phosphorylation in a time-dependent manner. ATP-induced phosphorylation of both residues was prevented by pre-application of the P2R antagonists, 300μM Suramin and 100μM PPADS (p-S783: p=0.037; p-S892: p=0.018 compared to ATP alone). Therefore, these results demonstrate that in astrocytes, P2Rs regulate the phosphorylation of S783 and S892 in GABABR2. In conclusion, our study suggests GABABR signalling in astrocytes is critically dependent upon the activation of P2Rs leading to the phosphorylation of key residues in the GABABR2. This synergistic interaction between P2Rs and GABABRs may act as a co-incidence detector to allow the fine-tuning of astrocytic Ca2+ signalling.