Human hepatocytes cultured with a hormonally defined medium on non-adherent poly-(2-hydroxyethyl methacrylate)-coated surface were able to form spheroids. The maintenance of liver-specific functions was assessed by following secretion of albumin, transferrin and alpha 1-antitrypsin that were still detectable after 4 months of spheroidal culture. Moreover, cytochrome P-450 IA was induced by methylcholanthrene for up to 2 weeks. This cell system is very promising for long-term in vitro studies of human hepatocyte functions.