Reaggregated occipital cortex cells of 19-day-old fetal rats were grown in a serum-free, chemically defined medium, and chronically exposed to impulse-blocking levels of tetrodotoxin (TTX) in order to study the role of bioelectric activity in synaptogenesis. As judged by phase-contrast microscopy, no differences were noticed in the development of neuronal networks in the TTX-treated vs control cultures. In addition, when TTX was withdrawn from experimental cultures at any stage of development, bioelectric activity qualitatively comparable to that of the control cultures appeared within 1 min. However, quantitative stereological EM analysis revealed a significant retardation in synapse formation and ultrastructural maturation of synaptic junctions during the first 3 weeks. Around 23 days in vitro, the central zone of the reaggregates in control cultures started to degenerate, but not earlier then day 27 in TTX-treated cultures. During this time, the control, but not the experimental cultures showed (in intact tissue regions mainly situated at the outside of the aggregates) a large and selective loss of spine synapses. It is concluded that functional blockade not only retards the early growth and maturation of synaptic networks but also prevents the later occurring selective loss of spine synapses.