We have previously reported that postnatal hypothalamic neurons can be maintained in low density culture using astrocyte conditioned medium. The present study was designed to establish a method for the culture of postnatal hypothalamic neurons in a chemically defined medium. Neurons were dissociated from the suprachiasmatic nucleus (SCN) of the hypothalamus of 21-day-old rats and plated on plastic dishes. First, the effects of several factors which have been known to exert trophic effects on neuronal cells were examined in culture medium containing 10% fetal bovine serum. We have found that platelet-derived growth factor, interleukin-1 beta and vitronectin in combination markedly increased the number of surviving neurons bearing processes. Next we tested such effects in serum-free minimum essential medium. When these factors were added together the SCN neurons could be maintained in culture for up to 3 weeks without medium change. In this supplemented medium, SCN neurons gradually extended processes from 3-5 days after plating, and the cell number with processes reached maximal at days 8-11. The cells were identified as SCN neurons by the immunocytochemical staining for microtubule-associated protein 2 (MAP2) and vasoactive intestinal polypeptide. This culture method may be valuable for investigating the electrophysiological properties and the mechanisms of regeneration of mature central neurons.