The occurrence of various cell-to-cell contacts and membrane specializations was quantitated in neocortical explant cultures prepared from 18-day-old rat embryos and exposed continuously to tetrodotoxin (TTX), gamma-aminobutyric acid (GABA) and an elevated level of magnesium ions (Mg++), respectively. Chronic TTX treatment resulted in a decrease in the number of synapses, paired neuronal membrane thickenings and nerve terminals with synaptic vesicles; the area of the neuronal compartment also decreased. By contrast, the gap junctions between glial cells increased, although the glial paired membrane thickenings decreased in number per unit area. Long-term GABA and Mg++ exposures did not alter significantly the occurrence of any of the cell contacts and membrane specializations analyzed when compared to control values. The results suggest an inhibitory effect of TTX on neuronal maturation and synapse formation in explant cultures of rat neocortex; this may lead secondarily to an increased demand for glial cell-to-cell communication.