Paracellular barrier properties of tissues are mainly determined by the composition of claudin heteropolymers. To analyze their molecular organization we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5 and -12 expressed in cerebral barriers were investigated. Tight junction strands were reconstituted by claudin-transfection of tight junction-free HEK293 cells. Homo- and heterophilic cis-interactions were analyzed by colocalization studies and fluorescence resonance energy transfer. The results indicate that cis-interactions and/or spatial proximity inside and outside of strands are ranked in the following order: Cld5/Cld5 > Cld5/Cld1 > Cld3/Cld1 > Cld3/Cld3 > Cld3/Cld5, no Cld3/Cld2. In addition, capability for homophilic trans-interaction showed claudin subtype-specific differences (as measured by means of enrichment at cell-cell contact). Heterophilic trans-interaction was analyzed in cocultures of differently monotransfected HEK293 cells and revealed defined compatibility. The ultra structure of reconstituted tight junction strands was investigated by freeze-fracture electron microscopy. A conserved tyrosine (corresponding to Y148 in mouse Cld5) in the extracellular loop 2 (ECL2) of classic claudins was found to be involved in formation of homo- and heteropolymeric tight junction strands as well as in paracellular tightening in vitro and in vivo. The data were used to generate a novel model of the molecular organization of tight junctions. Therefore, our data provide conceptual advances in the mechanistic understanding of the tightening of the paracellular cleft at the molecular level. This knowledge opens new strategies for pharmacological improvement of drug delivery.