Multiple forms of occludin were found in Madin-Darby canine kidney (MDCK) cells. In the absence of cell-to-cell contacts achieved by incubating cells in low-calcium growth medium, a cluster of lower-molecular-weight (LMW) occludin bands (approximately 65,000-68,000) was present in both MDCK I and II cells. On formation of tight junctions, achieved by changing the low-calcium growth medium to normal-calcium growth medium, a cluster of higher-molecular-weight (HMW) bands (approximately 72,000-75,000 for MDCK I cells and approximately 70,000-73,000 for MDCK II cells) was also expressed. The HMW occludin bands could be eliminated by phosphatase treatment. Therefore, the HMW forms of occludin appeared to be the hyperphosphorylated product of the LMW forms. These HMW forms were Triton X-100 insoluble, which correlated with their localization at the tight junctions. Furthermore, depletion of tight junction-localized occludin by an occludin extracellular domian peptide (20) correlated with a decrease in the HMW forms of occludin. In conclusion, phosphorylation of occludin may be a mechanism by which occludin localization and function are regulated.