Cytotactin is an extracellular matrix protein that is involved in neuron-glia adhesion and is found in both neural and nonneural sites. It is synthesized by glia but not by neurons. In this study, we have examined the binding of cytotactin to a variety of extracellular matrix components using uniform microscopic beads (Covaspheres) that could be labeled and then linked to purified molecules. Cytotactin-coated beads bound well to neurons, and this binding was strongly inhibited by anti-cytotactin antibodies but not by anti-neural cell adhesion molecule (anti-N-CAM) antibodies. In contrast, the binding of N-CAM-coated beads to neurons was inhibited by anti-N-CAM antibodies and not by anti-cytotactin antibodies. To identify a neuronal ligand for cytotactin, we tested several molecules for their ability to block the binding of cytotactin-coated beads to cells. A proteoglycan-containing fraction that copurified with cytotactin from brain extracts strongly inhibited binding, whereas neither a heparan sulfate proteoglycan from Engelbreth-Holm-Swarm tumor cells nor soluble cytotactin itself had a significant inhibitory effect. The neural proteoglycan also inhibited the binding of cytotactin-coated beads to fibroblasts. Digestion with chondroitinase, heparitinase, and hyaluronidase as well as immunological analyses suggested that the predominant species in the active fraction was a chondroitin sulfate proteoglycan with a Mr280,000 core protein bearing HNK-1 antigenic determinants and also indicated that hyaluronic acid was present in this fraction. In experiments on in vitro synthesis, it was found that the proteoglycan was synthesized in culture by embryonic chicken brain tissue but not by embryonic chicken glial cells. A series of binding experiments was performed on appropriately derivatized beads to confirm that the proteoglycan is a ligand for cytotactin and to check for the possibility that other extracellular matrix proteins might interact with one or the other member of this binding couple. Proteoglycan-coated beads and cytotactin-coated beads coaggregated readily. The aggregation was inhibitable by anti-cytotactin antibodies, soluble cytotactin, or soluble proteoglycan. Addition of laminin inhibited the binding of cytotactin-coated beads to proteoglycan-coated beads or to cells; this is consistent with data indicating that laminin interacts with a component of the proteoglycan-containing fraction. In contrast, fibronectin bound to cytotactin, but it did not bind to proteoglycan or interfere with the binding of cytotactin to proteoglycan. The results of this study are in accord with the idea that the functions of extracellular matrix components during neural and nonneural development may be modulated both by competition for shared cell surface receptors and by a network of molecular interactions among the matrix components themselves.