MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This aricle explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicines and cytochalasin B, on the degree of tightness of the occluding junctions. To study the degree of tightness, monolayers were prepared by plating MDCK cells on mylon disks coated with collagen. Disks were mounted as flat sheets between two Lucite chambers, and the sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers. Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation. Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane. Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell. Colchicine causes a profound disruption of microtubules but only a 27 percent decrease in the electrical resistance of the resting monolayers. Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance. The cortical ring of filaments remains in place but appears fragmented with time. We find that removal of extracellular Ca(++), which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca(++). Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible. The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTA-opened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.