Tight junctions are the most apical component of the epithelial junctional complex and are crucial for the formation and functioning of epithelial and endothelial barriers. They regulate selective diffusion of ions and solutes along the paracellular pathway and restrict apical/basolateral intramembrane diffusion of lipids. Research over the past years provided much insight into the molecular composition of tight junctions, and we are starting to understand the mechanisms that permit selective paracellular diffusion. Moreover, a complex network of proteins has been identified at tight junctions that is based on cytoskeleton-linked adaptors that recruit and thereby often regulate different types of signaling components that regulate epithelial proliferation, differentiation, and polarization.