Permeation of intercellular tight junctions in epithelia may be altered by maneuvers that affect the cytoskeleton. Conversely, agents that alter tight-junction permeability also often produce alterations in cytoskeletal structure. However, anatomic links between the tight junction and the cytoskeleton have not been clearly defined. We explore the anatomy of the perijunctional cytoskeleton by applying electron microscopy to cytoskeletal preparations of whole intestinal absorptive cells using detergent extraction techniques. Individual elements of the perijunctional cytoskeleton, including actin microfilaments as determined by S1 labeling, appear to associate with the tight junction by means of plaque-like densities that intimately associate with the lateral membrane at the site of the tight junction. Furthermore, such associations are not diffuse within the tight junction, but occur only at sites of fusions ("kisses") between lateral membranes that are thought to represent the specific intrajunctional sites at which the barriers to transjunctional permeation reside. These data provide evidence of intimate cytoskeletal-tight-junction associations, which may represent the anatomical basis for cytoskeletal control of tight-junction permeability.