It has been previously shown that adult mouse hepatocytes when co-cultured with rat liver-derived cell lines IAR-2 and IAR-20 can form organotypic hepatocyte islands consisting of cuboidal cells with expressed cell polarity and domain-specific localization of plasma-membrane proteins. The synthesis of alpha-fetoprotein, a fetal-specific antigen, was almost completely suppressed in these islands. It was noticed that organotypic islands were surrounded by fibrils and often covered with a roof of supporting cells.Here we demonstrate that the formation of adult mouse hepatocyte islands in co-cultures depends on the creation of 3D ECM scaffold formed by co-operative activity of hepatocytes and supporting partners. Noteworthy, the collagen type I network was observed exclusively around organotypic islands with complete suppression of alpha-fetoprotein synthesis.In contrast to non-transformed cells, IAR-2 cells, transformed either spontaneously or by transfection with mutant human N-ras A s n 1 2 cDNA, were completely unable to support in co-culture the differentiated morphology of hepatocytes and to suppress AFP synthesis. Transformed IAR-2 cells were able to produce most of the main ECM components except collagen type I, but failed to form extracellular fibrils both in pure culture and in co-culture with hepatocytes.Together, the data show that 3D ECM scaffold (possibly based on collagen type I) plays an important role in maintaining the differentiated morphology of hepatocytes and suppression of alpha-fetoprotein synthesis in vitro.