Stimulation of embryonic amphibian spinal neurons has been shown to produce calcium-dependent action potentials of long duration at early stages of development. These impulses become brief and sodium-dependent upon further differentiation. The neurons are now shown to exhibit spontaneous, transient elevations of intracellular calcium in culture during the early developmental period when activity produces greatest calcium influx. Removal of extracellular calcium during this period alone is sufficient to perturb differentiation, and influx through voltage-dependent calcium channels is shown to be required for standard development of neuronal phenotypes. No large changes in steady-state calcium levels occur in the cytoplasm during the maturation of cultured neurons despite a reduction of the calcium-dependent component of the impulse. Transient elevation of intracellular calcium is necessary for standard cytodifferentiation and may provide a link between electrical activity and gene expression.