In vivo, cellular relationships in the myenteric plexus are characterized by unusual compactness and by the arrangement of neurons and glia into ganglia and interconnecting strands. These features are lost when the myenteric plexus is placed in culture. In the present paper we test whether collagen type I, a major component of the matrix that surrounds the plexus in vivo, might have a role in maintaining normal neuron-glia relationships in this system. We report that a three-dimensional gel of rat tail collagen prevented the disaggregation of the guinea pig myenteric plexus in culture and induced the formation of a compact plexus-like cellular network when applied to disaggregated plexus cultures. These effects were not observed with soluble collagen. Immunohistochemical evidence was also obtained for synthesis of type I collagen by enteric glia. These observations indicate that type I collagen in a three-dimensional organization is capable of inducing and maintaining both the unusual compact organization of neurons and glial cells within myenteric ganglia and also the characteristic organization of these cells into an orderly network of ganglia and interconnecting strands.