Syntenin is an adaptor-like molecule that binds to the cytoplasmic domains of all four vertebrate syndecans. Syntenin-syndecan binding involves the C-terminal part of syntenin that contains a tandem of PDZ domains. Here we provide evidence that each PDZ domain of syntenin can interact with a syndecan. Isolated or combined mutations of the carboxylate binding lysines in the inter-betaAbetaB loops and of the alphaB1 residues in either one or both the PDZ domains of syntenin all reduce syntenin-syndecan binding in yeast two-hybrid, blot-overlay, and surface plasmon resonance assays. PDZ2 mutations have more pronounced effects on binding than PDZ1 mutations, but complete abrogation of syntenin-syndecan binding requires the combination of both the lysine and the alphaB1 mutations in both the PDZ domains of syntenin. Isothermal calorimetric titration of syntenin with syndecan peptide reveals the presence of two binding sites in syntenin. Yet, unlike a tandem of two PDZ2 domains and a reconstituted PDZ1+PDZ2 tandem, a tandem of two PDZ1 domains and isolated PDZ1 or PDZ2 domains do not interact with syndecan bait. We conclude to a co-operative binding mode whereby neither of these two PDZ domains is sufficient by itself but where PDZ2 functions as a "major" or "high affinity" syndecan binding domain, and PDZ1 functions as an "accessory" or "low affinity" syndecan binding domain. The paired, but not the isolated PDZ domains of syntenin bind also strongly to the immobilized cytoplasmic domains of neurexin and B-class ephrins. By inference, these data suggest a model whereby recruitment of syntenin to membrane surfaces requires two compatible types of bait that are in "synteny" (occurring together in location) and engages both PDZ domains of syntenin. The synteny of compatible bait may result from the assemblies and co-assemblies of syndecans and other similarly suited partners in larger supramolecular complexes. In general, an intramolecular combination of PDZ domains that are weak, taken individually, would appear to be designed to detect rather than drive the formation of specific molecular assemblies.