Tight junctions that tighten the paracellular cleft limit the drug permeability through the blood-brain barrier (BBB). The transmembrane tight-junction protein claudin-5 plays an essential role for the barrier integrity. Therefore, claudin-5-derived peptides were generated to characterize their function in opening the BBB. In vitro and in vivo, the peptides caused a transient permeability of substances that are typically not BBB-permeable while being well tolerated and not cytotoxic. Furthermore, the mechanisms of action as well as structural modelling have been investigated. Overall, the peptide-mediated penetration of drugs opens new specific treatment options for cerebral diseases. In contrast to claudin-5, Claudin-12 showed no function at the BBB and was further characterized in other tissues by using claudin-12 knockout mice.