An extracorporeal liver support system will require that liver cells maintain their normal differentiated function. This is more likely to be achieved utilizing a three-dimensional culture configuration rather than a simple monolayer culture. We present data on a human liver cell line attached and maintained on different three-dimensional supports, porous glass (Siran), silicon (Immobasil), and calcium-alginate beads. Albumin, fibrinogen, prothrombin, α1-acid glycoprotein and α1-antitrypsin secretions were measured. Proliferation was slower on each of the three-dimensional supports than on the monolayer culture. The protein secretion of all 5 proteins was highest in cells encapsulated in alginate; silicon beads supported greater protein secretion than glass. Cells on silicon or within alginate were rounded; those on glass grew in 2 configurations as flattened monolayers and as rounded colonies. Cells in alginate secreted as much protein as the whole liver (e.g., albumin, 14.88 g/1012 cells/day compared to the whole liver, ∼12 g/day). Three-dimensional culture of a human liver cell line leads to both proliferation and a high synthetic capacity, an important feature of cells suitable for an extracorporeal liver support system.