Most claudins are tight junction (TJ)-forming proteins. However, their interaction on the molecular level remains unresolved. It is hypothesized that the extracellular loops specify these claudin functions. It is assumed that the first extracellular loop (ECL1) is critical for determining the paracellular tightness and the selective paracellular ion permeability, and that the second extracellular loop may cause narrowing of the paracellular cleft. Using a combination of site-directed mutagenesis and homology modeling for the second extracellular loop (ECL2) of claudin-5, we found several amino acids important for claudin folding and/or trans-interaction to claudins in neighboring cells. These sensitive residues are highly conserved within one group of claudins, whereas the corresponding positions in the remaining claudins show a large sequence variety. Further functional data and analysis of sequence similarity for all claudins has led to their differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and nonclassic claudins (11-13, 16, 18, 20-24). This also corresponds to conserved structural features at ECL1 for classic claudins. Based on this, we propose a hypothesis for different pore-forming claudins. Pore formation or tightness is supported by the spatial encounter of a surplus of repulsing or attracting amino acid types at ECL1. A pore is likely opened by repulsion of equally charged residues, while an encounter of unequally charged residues leads to tight interaction. These considerations may reveal the ECLs of claudins as decisive submolecular determinants that specify the function of a claudin.