An in vitro system has been developed for the study of isolated hippocampal neurons from 18- or 19-day rat fetuses. Following trypsinization the cells are plated out at low density on polylysine-treated coverslips in an enriched medium. The isolated neurons rapidly attach to the substrate and initiate process extension. Little reaggregation occurs and the number of non-neuronal cells present is minimal. Unless co-cultured with tissue explants the neurons survive for only a few days; in the presence of hippocampal explants the initial growth of the isolated cells is improved and their survival in culture is extended to about two weeks. Some of the cells in such cultures develop a characteristic branching pattern closely resembling that of maturing hippocampal pyramidal cells in vivo. There is a clear relationship between the stage of the cells' development and their growth in culture. Cells which had completed DNA synthesis about 48 h before dissociation, and which were in the process of migration to the cortical plate, survived best in our cultures. Early post-mitotic cells which were still within the ventricular zone and cells which had already reached the cortical plate grew poorly. This system should permit the study not only of process formation by these cells, but also of their capacity to form specific synapses in vitro and of the biochemical constituents of their surfaces.