The paracellular barrier in epithelia and endothelia is formed by tight junctions (TJ). They limit and regulate the paracellular permeation of ions, solutes and proteins. The barrier properties of TJs in different tissues are mainly determined by the claudin composition of the heteropolymeric TJ-strands. To analyze the molecular organization of TJ, these strands were reconstituted by claudin-transfection of TJ-free HEK293-cells. A single claudin-construct was expressed to obtain defined homomeric TJ-strands. In addition, another claudin subtype or a member of the TJ-associated marvel domain protein family was expressed to obtain defined heteromeric TJ-strands. The ability to form strands and their morphology was investigated by freeze fracture electron microscopy or single molecule fluorescence nanoscopy. The barrier function of the TJ-strands was demonstrated by tracer exclusion imaging. cis-Interactions of claudin molecules were investigated by cellular fluorescence resonance energy transfer (FRET)- and native PAGE. trans-Interactions were detected by confocal cell-cell contact enrichment scanning. The mobility of claudins in the membrane was measured by fluorescence recovery after photobleaching (FRAP). This approach was used to characterize heterophilic compatibility of claudins. In addition, to elucidate mechanistic details of the claudin assembly, the blood brain barrier relevant Cld5 and Cld3 that differ in assembly properties were analyzed in more detail. Generation and investigation of Cld3/Cld5 chimeric mutants revealed protein segments and residues differentially involved in the assembly of claudin oligomers and TJ-strands. Futhermore, the domain III of Clostridium perfringens enterotoxin (cCPE), a high affinity ligand of a subset of claudins was used as a probe to gain information about the structure of the extracellular loop 2 of classic claudins. The experimental data was combined with schematic and molecular homology modeling of Cld3 and Cld5. The results provide novel molecular insights into subtype-specific assembly of classic claudins, paracellular barriers and the potential of pharmacological claudin-modulation, e.g. for paracellular drug delivery or treatment of claudin-overexpressing tumors.