To better understand the dynamics of the cellular processes involved in early neocortical development, we studied the neuritic differentiation and synaptogenesis of dispersed neurons grown in serum-free cultures under a wide variety of culture conditions. Microtubule-associated protein (MAP2), phosphorylated neurofilament (SMI 31) and synaptophysin immunocytochemistry was complemented with time-lapse studies. During the first week in vitro dissociated cortical neurons developed from roundish cells without processes to neurons with axons and differentiated dendrites, going through five distinct phases. The sequence of these phases was unaltered in a wide range of culturing methods, but the timing of the steps varied among cultures started with different cell densities. Synaptic terminals were first observed after 3-4 days in vitro, coincident with the beginning of dendritic differentiation. Synaptogenesis progressed at least until the end of the third week in vitro, despite a decline in cell density during the second week in vitro. The process of cellular differentiation of cerebral cortical neurons in vitro resembled the development of these cells in the intact tissue, suggesting that organized cell migration is not a prerequisite for the differentiation of single cortical neurons.