OBJECTIVE To establish primary cell culture conditions for tuberomammillary nucleus (TM) histaminergic neurons of neonatal rats. METHODS The tuberomammillary nucleus was dissected from the posterior part of the hypothalamus of neonatal rats, and neurobasal medium supplemented with B27 was used for the primary cell culture. The cultured neurons were identified by immunocytochemical method (ICC). RESULTS TM neurons from the hypothalamus of neonatal rats were successfully cultured under the experimental condition and tended to mature morphologically on the days 7 to 9 of culture. The cultured neurons were stained by ICC and the green immunoreaction product was seen in the neurons under the fluorescence microscope, indicating that the cultured neurons were histamine-positive. CONCLUSIONS TM neurons in the hypothalamus of neonatal rats can be cultured in vitro, and the primary cultured ones may serve as a cell model in vitro for the researches of central histaminergic system.