The morphological and biochemical changes that occur during in vitro neurogenesis of the mouse hypothalamus were studied in rotary cultures prepared from mice between 4 and 16 days of postnatal age. After 6 days of in vitro growth, histotypic cultures with a high degree of morphological differentiation were obtained in cultures prepared from 8- to 10-day-old mice. Before day 8, the cultures showed immature neurons, while after day 12 most of them exhibited an undesired number of degenerated cells. Light-microscopic, Golgi and ultrastructural studies clearly showed the stages of development of the neurosecretory cells in culture. The particular organization of the hypothalamic cells in these cultures can be homologized to its equivalent region in vivo, as demonstrated by their morphological similarities as well as by the fact that the majority of the neurons orient their axons toward the external part of the culture in order to release the neurosecretory material outside the in vitro grown neuronal population, as is the case in situ since hypothalamic neurons release their neurosecretory products at the vascular system. Biochemical parameters such as DNA, RNA and protein contents were determined during the period of in situ development used for the preparation of histotypic cultures and compared to the biochemical changes that occurred during in vitro maturation. The changes in the in vitro DNA and protein contents showed the same variation pattern as in situ. The DNA/protein and RNA/protein ratios also had comparable characteristics, having peak values at days 10 and 16 in situ and in the histotypic cultures prepared from 10-day-old mice. These studies have demonstrated the correlation between the in vitro biochemical and morphological development and the significance of the critical period during hypothalamic neurogenesis for successful organotypic preparations.