We have developed a system for the long-term cultivation of hypothalamic neurons at low density in defined serum-free medium, and have identified the major classes of cells in these cultures. Cells prepared from the dissociation of embryonic rat hypothalami are plated, in serum-free medium, onto monolayers of cortical astrocytes. Neurons adhere and begin to extend neurites soon after plating, and survive for up to several weeks in culture. Cell-type-specific immunological markers were utilized to identify neurons and the major classes of glial cells in these cultures. The culture of hypothalamic neurons in serum-free conditions provides a valuable system for the study of the cellular basis of hypothalamic neuronal heterogeneity and functional diversity.