We have studied interactions of tenascin with two chondroitin sulfate proteoglycans, neurocan and phosphacan. Neurocan is a multi-domain proteoglycan with a 136-kDa core protein that is synthesized by neurons and binds to hyaluronic acid, whereas the 173-kDa core protein of phosphacan, which is synthesized by glia, represents an extracellular variant of the receptor-type protein tyrosine phosphatase RPTP zeta/beta. Keratan sulfate-containing glycoforms of phosphacan (designated phosphacan-KS) are also present in brain. Immunocytochemical studies of early postnatal rat cerebellum demonstrated that the localization of neurocan, phosphacan, and phosphacan-KS all overlap extensively with that of tenascin, an extracellular matrix protein that modulates cell adhesion and migration. Binding studies using purified proteins covalently attached to fluorescent microbeads demonstrated that proteoglycan-coated beads co-aggregated with differently fluorescing beads coated with tenascin. The co-aggregation was specifically inhibited by Fab' fragments of antibodies against tenascin or the proteoglycans and by soluble neurocan, phosphacan, and tenascin. A solid phase radioligand binding assay confirmed that neurocan, phosphacan, and phosphacan-KS bind to tenascin but not to laminin and fibronectin. Chondroitinase treatment of the proteoglycans or addition of free chondroitin sulfate had no significant effect, indicating that the binding activity is mediated largely via the core glycoproteins. Scatchard analysis demonstrated high affinity binding of 125I-phosphacan, phosphacan-KS, and neurocan to a single site in tenascin, and neurocan and various glycoforms of phosphacan all inhibited binding of 125I-phosphacan to tenascin. In studies of cell adhesion to proteins adsorbed to Petri dishes, phosphacan inhibited adhesion of C6 glioma cells to tenascin whereas neurocan had no effect. Our results suggest that tenascin binds phosphacan and neurocan in vivo and that interactions between chondroitin sulfate proteoglycans and tenascin may play important roles in nervous tissue histogenesis, possibly by modulating signal transduction across the plasma membrane.