The extracellular matrix (ECM) promotes the differentiation of many cell types, and ECM remodeling in the liver has been implicated in embryonic development, tissue injury, and oncogenesis. Integrins are heterodimeric ECM receptors that play critical roles in transducing the composition of the ECM in the cell environment. We previously showed that mouse H2.35 cells, a conditionally transformed, liver-derived cell line, assume a more differentiated hepatocyte morphology and enhanced liver-specific gene expression when the cells are cultured on gelatinous ECM substrata. Here we show that H2.35 cells express relatively high levels of α3β1-integrins, similar to that previously shown for immature hepatocytes, transformed hepatocytes, and biliary cells. However, the cell morphological responses that depend on α3β1-integrin have not been defined. We found that transfecting H2.35 cells with antisense RNA construct directed to α3-subunit messenger RNA perturbs the initial cell attachment to laminin and collagen, and strongly inhibits cell morphological, proliferative, and gene expression responses to a collagen gel substratum. In situhybridization to mouse embryo tissues demonstrates the presence of α3-subunit messenger RNAs in newly formed hepatocytes. We suggest that α3β1-integrins are important for immature and transformed hepatocytes to respond morphologically to the extracellular matrix