A compartmented culture system in which distal neurites from newborn rat sympathetic neurons entered a fluid environment separate from that bathing the cell bodies and proximal neurites was used to investigate effects of extracellular Ca2+ deprivation on nerve fiber growth. Neurites readily grew into, elongated for many days within, and regenerated after neuritotomy within distal compartments substantially deprived of Ca2+ (0 added Ca2+, 0.5-5 mM EGTA), provided Ca2+ was supplied to the cell bodies. The Ca2(+)-deprived neurites generally extended at rates 20%-35% slower than controls. Growth of neurites did, however, cease within 2 days when the cell bodies were deprived of Ca2+, and the neurites and cell bodies eventually degenerated. These results show that neither extracellular Ca2+ nor the influx of Ca2+ at or near the growth cone is required for sustained neurite growth. They also rule out the possibility that the promotion of neurite growth by nerve growth factor is mediated, by the influx of extracellular Ca2+.