The development of the action potential and responses to neurotransmitters have been described for a population of embryonic spinal neurons developing in vivo. A comparable pattern is seen for spinal neurons developing in dissociated cell culture. The impulse appears very early in this developmental sequence, and the action potential involves a large inward Ca2+ current. Since Ca2+ is a ubiquitous intracellular regulator, we questioned whether a large influx of Ca2+ is necessary for the subsequent differentiation of membrane properties. Embryonic Xenopus neurons grown in normal culture medium do not make Ca2+- or Na+-dependent action potentials in their cell bodies in a Ca2+-free saline containing tetrodotoxin (TTX). To achieve a chronic blockade of impulse activity, neurons were grown in a medium in which Ca2+ was replaced by Mg2+, and to which 1 mM EGTA was added. In some instances TTX was present. Neurons grown in these experimental culture media extend neurites more rapidly than controls. Action potentials cannot be elicited from neurons when examined in experimental medium. However, examination in saline reveals that the change in the ionic dependence of the impulse is indistinguishable from that observed in neurons grown in normal medium. Furthermore, the time of onset of responses to GABA is unaffected by this experimental treatment. Thus the expression of Ca2+- and Na+-dependent action potentials seems not to play a part in the early differentiation of these membrane properties. However, the later development of GABA sensitivity is reduced.