WIF-B9 is a suitable model for in vitro studies of hepatocyte polarity. To better understand polarity establishment, we have localized key proteins of the adhesion system, cytoskeleton, and tight junctions soon after plating, when most cells are isolated or in doublets. In isolated attached cells, only cytoskeletal proteins (tubulin, cytokeratins) displayed a precise localization. As soon as two cells formed a doublet, E-cadherin, -, -, and -catenins, and p120 protein were present at the doublet contiguous membrane. Actin, ezrin, and zonula occludens-1 (ZO-1) colocalized at this membrane, but not in all doublets: ezrin was present only at contiguous membrane expressing ZO-1, and ZO-1 was present only at membrane expressing actin. In contrast, occludin was spread throughout the doublet cytoplasm. With time in culture, these proteins localized transiently, as in cells expressing simple epithelial polarity, and finally, as in hepatocytes. We conclude that during WIF-B9 early polarization, key proteins are settled according to a hierarchy, as has been shown for Madin-Darby canine kidney cells. Cytoplasmic complexes of E-cadherin-catenin were detected during the whole polarization process; they were more abundant in fully polarized cells.