Studies were carricd out to analyze how different extracellular matrix (ECM) moleculcs regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non-adhesive plastic dishes that were pre-coated with defined densities of eithcr laminin, fibroncctin, type I collagen, or type IV collagcn. Sparse cell plating densities were used to minimize cell-cell contact formation and a11 studies were carried out in chemically defined medium that contained a 5aturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver-specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell sprcading, cxpression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down-regulation of diffcrcntiated functions and again, dishes coated with all four types of ECM molecules produccd similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.c., between different gcnctic programs) is not limited to a single ECM molecule, a distinct thrre dimensional ECM geometry, or due to alteration of cell-cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading. o 1'192 WiIq-Liss. Inc