ATP released from synaptic terminals serves as potent neuromodulator within hypothalamic structures concerned with the central regulation of body temperature and modulation of the pyrogen-induced fever reaction in the rat (Gourine et al., 2002, 2005). At the level of the median preoptic nucleus (MnPO), ATP and its break-down product adenosine (ADO) are possibly involved in the final steps of fever induction in mammals. The aim of the present study was to characterize purinergic receptor signalling in rat MnPO-intrinsic cells. A primary culture system of the MnPO was established from topographically excised brain tissue of 5–6 days old rat pups. The Fura-2 ratio imaging technique was used to quantify purinoceptor-induced calcium signalling in a MnPO microculture, stimulated at RT by superfusion with various agonists for 2PX (ATP, 2Me-SATP, a,ß-Me-ATP, Bz-ATP) and ADO (NECA, CCPA) receptors as well as 2Me-SATP in the presence of various antagonists (PPADS, TNP-ATP, RB) or zinc ions. Identification of differentiated MnPO cells in the culture was performed by immunolabeling with cell-type specific antisera. Expression of 2PX receptor subtypes within the MnPO was investigated using real-time PCR and immunocytochemistry. Concentrations of the proinflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) released into the supernatant of ATP- and LPS-stimulated MnPO microcultures were determined by specific bioassay. Both ATP and 2Me-SATP (0.1 – 1 mM/L) evoked an increase in [Ca2+]i upon superfusion stimulation in MnPO neurons, astrocytes and oligodendrocytes, with calcium entering the cell from the extracellular space. However, a,ß-Me-ATP and Bz-ATP (1 - 10 mM/L), the latter specific for the P2X7 receptor subtype, as well as the ADO analogues NECA and CCPA, specific for P1 receptors, proved to be ineffective in eliciting intracellular calcium signalling in any cell type. The P2X-specific antagonists PPADS, TNP-ATP and Rb2 (1 - 10 mM/L) were able to signficantly reduce the peak increment of [Ca2+]i evoked by 2Me-SATP (100 nM/L). Zinc ions present in the superfusion medium augmented activation exclusively of neurons by ATP, indicative of 2PX2 purinoceptor prevalence in neurons. Real-time PCR revealed marked expression of the 2PX2 > 2PX4 > 2PX7 purinoceptor subtypes in the neonatal rat MnPO. Incubation of primary MnPO cultures with ATP caused dose-dependent release of TNF-a and IL-6. The marked release of both cytokines due to stimulation with lipopolysaccharide (LPS) from gram-negative bacteria proved to be significantly reduced in the presence of the P2X receptor antagonist PPADS and even more so TNT-ATP. Immunocytochemical analysis of primary MnPO cultures in combination with the real-time PCR data suggests P2X4 mediated activation of microglial cells and/or astrocytes as targets for both endogenous ATP and possibly also LPS-stimulated ATP secretion.