When Xenopus spinal cord (SC) neurons are grown on an appropriate substrate of basal lamina molecules, the agrin they externalize along their neuritic outgrowth remains bound to the substrate even after the neurons are removed. Here we demonstrate that these former neuritic pathways containing substrate-bound, neural agrin cause an accumulation of acetylcholine receptors (AChR) and cholinesterase (ChE) at sites of contact with muscle cells and inhibit AChR aggregation over the rest of the muscle cell surface. These local and global synaptogenic effects were not triggered by former neuritic pathways that were agrin-negative. The length of AChR accumulation along the agrin pathways contacted by individual muscle cells corresponded to a saturation process, in agreement with the notion that muscle cells have a limited capacity to cluster AChR. The AChR accumulation caused by the agrin pathways was almost twice as extensive as that induced by living neurites. It is concluded that agrin and possibly other synaptogenic molecules externalized by competent SC neurons bind to the culture substrate in quantities which are more than sufficient to account fully for the local and global changes in AChR and ChE distribution associated with embryonic nerve-muscle synaptogenesis.