Principal neurons from the superior cervical ganglia of rat fetuses were maintained for up to 101 days in dissociated cell cultures in a serum-free, chemically-defined medium; non-neuronal cells were killed by the periodic addition of fluorodeoxyuridine to the medium. Intracellular recordings, obtained at various times between 16th and 98th day in vitro, showed that these neurons could generate substantial (up to 90 mV) action potentials in response to depolarizing current injections; these responses were dependent on tetrodotoxin-sensitive Na+ channels, cobalt-sensitive Ca++ channels, and tetraethylam monium-sensitive K+ channels. Action potentials were often followed by prominent, long hyperpolarizing after-potentials (10-15 mV, > 150 ms); the duration of these after-potentials was reduced by the addition of Co++ (2-5 mM) to the perfusate. Acetylcholine depolarized these neurons by a hexamethonium-sensitive mechanism. To determine whether sympathetic neurons formed synapses in a defined medium, intracellular recordings were obtained from pairs of neighboring neurons. Synaptic interactions were frequently observed at all times in vitro (up to 60% of all pairs tested). At many synapses, both hyperpolarizing and depolarizing DC potential changes spread from one neuron to another. At other synapses, the spread of DC potential changes could not be directly demonstrated; however, interactions at such synapses were not inhibited by antagonists of several neurotransmitters, by elevation of the Mg++/Ca++ ratio, or by the addition of Co++. Thus most, if not all, of the synaptic interactions among sympathetic neurons were electrotonic; such electrical synapses were not observed among dorsal root ganglion neurons maintained in the same medium. These data indicate that, when maintained in a chemically-defined culture medium, sympathetic neurons of rat fetuses express many of the basic membrane properties observed in neurons of superior cervical ganglia recently removed from adult rats. However, fetal sympathetic neurons maintained in this defined medium also differ from their counterparts in vivo; they adopt a mode of synaptic transmission (electrical) that has not been observed in the sympathetic ganglia of the adult rat. Thus, as late as the 21st embryonic day, not only the choice of neurotransmitter, but also the mode of transmission has not been irrevocably determined in sympathetic neurons.