Culture thickness, numerical density of neurons and neuronal survival were studied in timed series of control and tetrodotoxin-silenced neocortical cultures to provide information on the role of bioelectric activity on neuronal development. In control cultures, culture thickness and number of surviving neurons decrease during the first weeks in vitro, but remain constant between 2 and 3 weeks indicating that the cultures are essentially mature. In the 4th week in vitro a further decrease in surviving neurons was observed. In tetrodotoxin-treated cultures the number of surviving neurons decreased significantly between 1 and 2 weeks in vitro, to remain constant thereafter. However, culture thickness significantly increased at 3 and 4 weeks in vitro after an initial drop between 1 and 2 weeks. Compared to age-matched controls at 2 and 3 weeks in vitro, only ca 50% of the neurons survived the loss of bioelectric activity. Similar differences were present between 1 and 2 weeks. Thus, the loss of all measurable bioelectric activity induces neuronal death in neocortical explants, but promotes neuropil formation by the surviving cells.