The proliferative status and time of origin of dissociated cells from cerebral cortex, basal ganglia, diencephalon, mesencephalon and rhombencephalon of the fetal rat brain have been analyzed. The distributions of cells in different phases of the cell cycle after dissociation and after 7 days in culture have been determined with flow cytofluorometry. Two separate DNA indicators, propidium iodide and Hoechst 33258, have been used. Almost all of the dissociated cells recovered from the 5 brain regions, between embryonic days 15 and 20, are in G1, or growth arrest phase of the cell cycle. In contrast, only about 55% of the liver cells (same fetal age) are in G1, or growth arrest phase. The times of the last in vivo mitoses of the dissociated cells have been determined by maternal injection of [3H]thymidine and autoradiography of cultures. The majority of the cells recovered on embryonic days 16 and 17 from the 5 brain regions appeared to have undergone DNA synthesis within a period of 24 h prior to dissociation. When the fetuses were sacrificed 96 h after the injection, less than 20% of the cells were labelled, and grain density was drastically reduced. Most labelled cells survive in the serum-free culture medium for more than 7 days. Dissociated cultures of synchronized and birth-dated cells from different brain regions of fetal rat should prove particularly useful for the study of cellular development and function.