A primary culture system of cells derived from two circumventricular organs (CVO) of the rat brain was established. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) were dissected from the rostral wall of the third ventricle and its cells taken into culture after mechanical dissociation. The cells were cultured in a modified microculture chamber system ensuring relatively high cell density despite their low absolute number. When animals were injected with Evans blue prior to cell preparation, the macroscopically visible penetration of the dye into the parenchyma of the CVOs could be used as guidance during tissue isolation and labelled cells could be identified in culture. Cultured CVO neurones and astrocytes were identified using antibodies against cell type specific marker proteins. The histochemical NADPH-diaphorase staining was used for the detection of nitric oxide synthase in tissue sections of both CVOs and in their cultured neurones. In addition, angiotensin II (ANG II)-evoked elevations of the intracellular Ca2+ concentration ([Ca2+]i) in single cultured OVLT neurones were measured. The described methods will be useful for further characterization of CVO neurones and astrocytes.