We studied the expression, distribution, and phosphorylation of the tight junction (TJ) protein occludin in confluent MDCK cell monolayers following three procedures for opening and resealing of TJs. When Ca(2+) is transiently removed from the culture medium, the TJs open and the cells separate from each other, but the occludin band around each cell is retained. When Ca(2+) is reintroduced, the TJs reseal. When the monolayers are exposed to prolonged Ca(2+) starvation the cells maintain contact, but occludin disappears from the cell borders and can be detected only in a cytoplasmic compartment. When Ca(2+) is reintroduced, new TJs are assembled and the transepithelial electrical resistance (TER) is reestablished in about 20 hr. Monolayers treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) show a different pattern of TJ opening: the cell-cell contact is maintained but the TJ strand network, as seen in freeze-fracture replicas, becomes discontinuous. Occludin is still localized at the cell periphery, but in a pattern of distribution that matches the discontinuous TJ. These TJs do not reseal even 24 hr after removal of the TPA. Western blot analysis showed that the 62-65 kD double band of occludin did not change with these treatments. However, in vivo phosphorylation analysis showed that the TPA treatment reduced the phosphorylation levels of occludin, while the prolonged Ca(2+) starvation completely dephosphorylated the two occludin bands. In addition, a highly phosphorylated 71 kD band that immunoprecipitates with occludin is not present when TJ is opened by the Ca(2+) removal. Phosphoaminoacid analysis showed that the 62-65 kD occludin bands are phosphorylated on serine and threonine, while the 71 kD band was phosphorylated exclusively on serine. Our results provide further evidence that phosphorylation of occludin is an important step in regulating TJ formation and permeability.