Growth of neurites from trypsin-dissociated retinal neurons from chick embryos is very sensitive to the extracellular calcium concentration. Blockers of calcium permeability such as Co2+, Mn2+, La3+ and nitrendipine, when added to dissociated neurons, decrease the fraction of cells that extend neurites and the rate of neurite growth without influencing cell-substratum adhesion or survival capacity. Inhibition is concentration dependent, is related to the age of the donor chick embryo and can be prevented by increasing the extracellular calcium concentration. 50% inhibition in 8-day neurons is produced by 120 microM Co2+, 250 microM Mn2+, 50 microM La3+ and 10 microM nitrendipine in medium containing 1.8 mM Ca2+. Inhibition of neurite extension is accompanied by a concentration dependent inhibition of synapse formation between retinal neurons and muscle cells in culture, as determined by intracellular recording. 50% inhibition in the fraction of innervated myotubes is produced by 0.4 mM Co2+ and 4 mM Mn2+. These results suggest that: (1) a voltage-dependent calcium flux is a signal not only for growth cone expansion but also for neurite extension in primary dissociated neurons; and (2) that neurite extension is a prerequisite for synaptogenesis between neurons and muscle cells in culture.