A three-chamber culture system was devised in which neurites growing from small clusters of somas of sympathetic neurons penetrated a virtually fluid-impermeable barrier; thus the local fluid environment of the distal portions of the neurites could be controlled independently of the local fluid environment of the somas and proximal portions of the neurites. Neurites regularly penetrated the barriers if a high concentration of nerve growth factor was present on both sides, but never penetrated into chambers to which no nerve growth factor had been added. After neurites crossed the barrier, local removal of nerve growth factor from the distal portions of the neurites caused the growth of these portions to stop, and they eventually appeared to degenerate even though nerve growth factor was continuously present in the chamber that contained their somas and proximal portions. In contrast, local nerve growth factor was not required at the somas and proximal portions of the neurites; many neurons survived its withdrawal provided their somas were associated with neurite bundles that crossed into a chamber containing nerve growth factor. These results show that the growth, and probably the survival, of neurites depends upon nerve growth factor in their local environment, regardless of the nerve growth factor concentrations to which other portions of the neuron are exposed. This is entirely consistent with the notion that nerve growth factor released by sympathetic target tissues promotes the establishment and maintenance of appropriate neuron-target connections during development.