Astrocytes provide neurones with metabolic and structural support. There is also evidence that these numerous glial cells may also play an active role in controlling neuronal excitability, synaptic transmission, plasticity and, therefore, information processing. Astrocytes residing in different parts of the central nervous system are tuned to sense local microenvironment, including neurochemical changes associated with enhanced activities of local and projecting neurones. Previous studies demonstrated that astrocytes show elevations in intracellular [Ca2+] in response to 5-HT (1-3). In this study we aimed (i) to determine whether hippocampal, cerebellar and brainstem astrocytes are equally sensitive to 5-HT; (ii) to identify the receptor(s) which mediate the effects of 5-HT on astrocytes residing in different parts of the CNS. Primary astroglial cultures were prepared from the hippocampus, cerebellum and the brainstem tissue of neonatal rats (P2-3). After 6 days in culture, cells were loaded with a conventional Ca2+ indicator Fura-2 and changes in intracellular [Ca2+] evoked by 5-HT (10 µM) were recorded using fluorescent microscopy. 5-HT receptor agonists and antagonists were used to characterize the identity of 5-HT receptors which mediate the recorded effects of serotonin. Astrocytes cultured from all three brain regions responded to 5-HT with vigorous elevations in intracellular [Ca2+] (EC50 7-10 µM). Phenylbiguanide (PBG, 5-HT3 agonist) in concentrations 0.1-0.3 µM mimicked the effects of 5-HT (induced increases in [Ca2+]i) in hippocampal astrocytes. PBG had no effect on [Ca2+]i in the brainstem or cerebellar astrocytes. 5-HT-evoked [Ca2+]i responses in hippocampal astrocytes were completely blocked by 5-HT receptor antagonist granisetron (20 µM), suggesting that only 5-HT3 receptors are expressed by these cells. [Ca2+]i responses elicited by 5-HT in cerebellar and brainstem astrocytes were blocked by phospholipase-C (PLC) inhibitor U73122 (5 µM), suggesting that 5-HT2 receptors might be involved. The 5-HT2A antagonist ketanserin (0.01-1 µM) blocked 5-HT-induced [Ca2+]i responses in both cerebellar and brainstem astrocytes (but not in hippocampal astrocytes). Neither 5-HT2C agonist WAY161503 (0.01 – 5 µM) nor 5-HT2B agonist BW723C86 (0.001 – 1 µM) had an effect on [Ca2+]i in astrocytes of any of three brain regions studied. These data indicate that astrocytes residing in different parts of the central nervous system express different functional 5-HT receptors. These differences are retained in a highly reduced culture conditions. Hippocampal astrocytes express ionotropic 5-HT3 receptors, while cerebellar and brainstem astrocytes express G protein-coupled 5-HT2A receptors. These data imply that 5-HT actions in the CNS may (at least in part) be mediated via the actions of this transmitter on 5-HT3 and 5-HT2A receptors expressed by astroglia.