Recent experiments have indicated that neural agrin is deposited at newly forming nerve-muscle synapses and has a primary synaptogenic role there. As a step toward assessing how the spatial arrangement of new synaptic sites is regulated, we compared the pattern of agrin deposition by Xenopus neurites on culture substrate and on muscle cells. The neurons were grown on a substrate that bound their externalized agrin so tightly that it remained bound even when the neurites retracted spontaneously or were eliminated experimentally. By contrast, the neural cell adhesion molecule, NCAM, was not left behind on the substrate when the neurites were eliminated. Agrin, visualized by immunofluorescent staining, was deposited on the culture substrate in a continuous fashion along virtually the entire neuritic arbor of many spinal cord (SC) neurites. The pattern of agrin deposition by the same neurites changed from continuous to discontinuous when the neurites contacted muscle cells, and it became continuous again when the neurites returned to the culture substrate. The sites of agrin deposition on muscle cells were also sites of accumulation of ACh receptors (AChRs). Dorsal root ganglion (DRG) neurons and some SC neurons did not deposit agrin along their neuritic outgrowth, either on the culture substrate or on the muscle cells, and did not induce AChR accumulation at sites of contact with muscle cells. Besides adding to the evidence in support of agrin's synaptogenic role, the findings indicate that muscle cells significantly influence how neural agrin and synaptic sites become distributed along paths of neurite-muscle contact.