Apelin-12 and its G-protein coupled receptor APJ have been described to play a physiological role as a neuromodulatory system in the hypothalamic control of body fluid and temperature homeostasis. Both the parvocellular paraventricular nucleus (pPVN) and the median preoptic nucleus (MnPO) of the rat hypothalamus are known to act as neuroglial relay stations for the perception of afferent signals transducing information from central and peripheral thermo- or osmosensors. Hypothalamically released nitric oxide synthesized by neuronal nitric oxide synthase (nNOS) represents another major neurotransmitter system within both the osmo- and thermoregulatory circuitries. The aim or the present study therefore was to evaluate apelinergic innervation of nNOS-positive and –negative neurons within the pPVN and MnPO, as well as apelin-12 induced cell signalling in MnPO neurons and astrocytes. Employing immunohistochemical analysis in perfusion-fixed coronal rat brain sections (20 mm) cut in a cryostat at -20°C, hypothalamic APJ receptor expression could be localized preferentially in the anterior hypothalamic area, the parvocellular (pPVN) and magnocellular PVN (mPVN), as well as the (ventro-)medial preoptic area (MPA, VMPO). In addition, all subunits of the MnPO, the anteroventral third ventricular region (AV3V) and the Organum vasculosum laminae terminalis (OVLT) strongly expressed the APJ protein. These neuroglial structures are notably known to be involved in central body fluid (AV3V, mPVN, MnPO, OVLT) and core temperature (MPA, VMPO, MnPO, OVLT) homeostasis. While in most hypothalamic structures including the MnPO, immunoreactive APJ expression was restricted to plasma membranal or synaptic patches, cytosolic location of the APJ protein in all subunits of the PVN enabled co-localization studies with nNOS. Within the pPVN and the mPVN, some 22% respectively 57% of all nitrergic neurons were also APJ-immunopositive, indicating apelinergic regulation of PVN NO-producing neurons. To study apelin-12 induced cellular activation within the AJP-endowed MnPO, a primary microculture system was established from topographically excised brain tissue of 5–6 days old rat pups. The Fura-2 fluorescence ratio imaging technique was used to quantify apelin-12 induced calcium signalling in MnPO cells superfused with oxygenated HEPES buffer containing apelin-12 (10^-6 M/L), glutamate (10^-6 M/L) or both receptor agonists together. Only a very limited number of MnPO neurons and astrocytes responded to apelin-12 stimulation with an increase in intracellular calcium concentration ([Ca²⁺]i). On the other hand, glutamatergic activation of MnPO cells, possibly reflecting afferent thermal input, was markedly reduced in the presence of apelin-12, with fluorescence Dratio values representing [Ca²⁺]i decreasing from 0.20 0.12 to 0.03 0.04 for neurons (n=9) and from 0.13 0.04 to 0.03 0.03 for astrocytes (n=7).