Liver-specific functions were examined in two novel human hepatocyte lines established from normal liver tissue (HH29, HHY41) and one human hepatoma line (Hep G2), cultured either in a collagen gel sandwich configuration or on plain plastic dishes. At confluence, levels of albumin and alpha-fetoprotein mRNA were markedly increased in all lines cultured in the collagen gel sandwich over those on plastic, while levels of mRNA for mitochondrial elongation factor (EFTU), a ubiquitous enzyme, were equivalent. Secretion of albumin by cells cultured in the collagen gel sandwich was significantly increased in parallel with the abundance of albumin mRNA. Hepatocytes in the collagen gel sandwich configuration secreted alpha-fetoprotein of a higher molecular weight in addition to alpha-fetoprotein of standard molecular weight. The three-dimensional configuration of the collagen gel sandwich enhances the expression of liver-specific functions of human hepatocytes from continuous cell lines, probably by preserving proper cellular polarity in vitro.