Occludin’s role in mammalian tight junction activity was examined by ‘labeling’ the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cells with the Lac repressor gene. Hyg^R clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting Hyg^R/Neo^R clones were plated on porous inserts and allowed to form tight junctions. Once steady state transep-ithelial electrical resistance was achieved, isopropyl-β-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca²⁺ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also colocalized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl-β-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transep-ithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl-β-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densit-ometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca²⁺ medium were treated with isopropyl-β-D-thiogalactoside for 24 or 48 hours, before Ca²⁺ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175±11 nm to 248±16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl-β-D-thiogalac-toside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.