The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitro and in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton.