AbstractWe previously reported the expression of ZO‐1 in cell types that do not form tight junctions. Here we compare the molecular environments of ZO‐1 in epithelial cells, primary cultures of astrocytes and in the non‐epithelial S180 sarcoma cell line. ZO‐1 co‐localizes with a subset of actin filament in all cell types. In astrocytes, ZO‐1 is found concentrated in discrete bands at points of cell‐cell contact. Indirect immunofluorescent microscopy shows that these bands of ZO‐1 co‐localize with the adherens junction proteins vinculin and α‐actinin, and with the antigen recognized by a pan‐cadherin antibody. In contrast, ZO‐1 in S180 cells, which exhibit limited cell‐cell interactions, is diffusely distributed over the plasma membrane, with concentrations in lamellipodia where actin filaments accumulate. ZO‐1 does not co‐localize with vinculin at focal adhesions in this cell type. Analysis of ZO‐1 immunoprecipitation profiles from different cell types, performed under conditions previously demonstrated to maintain interactions between ZO‐1, ZO‐2 and p130 from the MDCK epithelial cell line, show that the proteins which co‐precipitate with ZO‐1 vary with cell type. Precipitation of polypeptides at 165 kDa, potentially ZO‐2, and 65 kDa occurs in both a mouse kidney tubule epithelial cell line and the non‐epithelial S180 cells. No proteins specifically associate with ZO‐1 immunoprecipitated from astrocytes. Spectrin, α‐actinin, vinculin and cadherin are not detected in immunoblots of ZO‐1 immunoprecipitates from any cell type. © 1995 Wiley‐Liss, Inc.