High yields of viable human hepatocytes were obtained by enzymatic perfusion of the left hepatic lobe of kidney donors and cultured alone or with an epithelial cell line derived from rat liver. In conventional cultures, human hepatocytes did not survive more than 2 to 3 weeks and by Day 8 decreased their ability to secrete albumin. When co-cultured, they survived for more than 2 months and secreted high levels of albumin even in a serum-free medium. This long-term survival appeared to correlate with production of an extracellular material which is rich in Type III collagen. In vitro phenotypic alterations of parenchymal cells were reversed by addition of rat liver cells and were characterized by recovery of cuboidal morphology, increased albumin secretion and a shift from Type I to Type III collagen deposition. Rat liver epithelial cells could not be replaced by nonhepatic epithelial cells. These observations suggest that when adult human hepatocytes are maintained in a culture which closely resembles their in vivo environment, they are capable of continuing to actively express specific cell functions.