The extracellular matrix plays an important role for maintaining liver functions. We examined the effects of type I collagen and fibronectin on the expression of liver-specific genes in rat primary hepatocytes. When primary culture hepatocytes were overlaid with a type I collagen-gel, the expression of liver-specific genes (tyrosine aminotransferase, aldolase B, and albumin) increased by 4–5 times, compared with not overlaid hepatocytes. In contrast, the expression of non-liver-specific genes (GAPDH and β-actin) was suppressed under the same conditions. The addition of fibronectin together with type I collagen-gel further enhanced the expression of liver-specific genes by 1.4–1.8 times. The addition of GRGDS peptide instead of fibronectin with the collagen-gel had a similar effect on hepatic gene expression to that of fibronectin. Addition of fibronectin alone exhibited had no effect on gene expression. These results suggest that type I collagen and fibronectin synergistically induce liver-specific genes.