The integrity of the tight junction barrier in epithelial and endothelial cells is critical to human health, but we still lack a detailed mechanistic knowledge of how the barrier is formed during development or responds to pathological and pharmacological insults. This limits our understanding of barrier dysfunction in disease and slows the development of therapeutic strategies. Recent studies confirm the long-maintained but previously unsupported view that the zonula occludens (ZO) proteins ZO-1 and ZO-2 are critical determinants of barrier formation. However, ZO proteins can also be components of adherens junctions, and recent studies suggest that ZO proteins may also promote the assembly and function of these junctions during epithelial morphogenesis. We review these studies and outline several recent observations that suggest that one role of ZO proteins is to regulate cytoskeletal dynamics at cell junctions. Finally, we propose a model by which the functional activities of ZO proteins in the adherens junction and tight junction are differentiated by a novel regulatory motif known as the U6 or acidic motif.