Alpha-dystrobrevin (a-DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex in skeletal muscle cells. Isoforms of a-DB show different localization in cells and tissues, at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, a-DBs are assumed to be important in cell signalling and cell differentiation. We have primarily assessed the role of a-DB in two epithelial cell lines (MDCK I, HT 29), which represent different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-a-DB antibody, we have investigated its expression, localization and association with tight junction (TJ)- associated proteins (ZO-1, occludin) before and after protein kinase C (PKC) activation with phorbol myristate acetate. Distinct subsets of a-DB isoforms were detected in the two cell lines by immunoblotting. In both cell lines there was submembranous localization of a-DB both apically and basolaterally, shown with confocal imaging. PKC activation caused a reorganization of TJ, which was parallel to increased localization of a-DB to TJ areas, most pronounced in MDCK I cells. Moreover, actin and ZO-1 co-immunoprecipitated with a-DB, as displayed with immunoblotting. Our findings suggest that a-dystrobrevin specifically is associated with the tight junctions during their reorganization. © Springer Science+Business Media, Inc. 2005.